Name: evaluation of the efficacy and toxicity of rnas targeting hiv-1 production for use in gene or drug therapy
Uploaded: 2016-09-05T14:00:00.000+00:00
Duration: 12 min 3 s
Description: Watch this Scientific Journal Video about Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy at JoVE.com

ここで紹介する作品は、（AGにDCB-120266、PPP-133377およびHBF-348967を付与）健康研究（CIHR）のカナダの研究所によってサポートされていました。

1.細胞およびトランスフェクション<ol><li>ダルベッコ改変イーグル培地（DMEM）中で培養HEK 293T細胞を、10％ウシ胎児血清（FBS）および1％ペニシリン/ストレプトマイシンを補充しました。細胞培養培地中の2×10 5細胞/ mlの懸濁液を調製します。それぞれ、ウイルス産生のために、24ウェル、96ウェル、12ウェルプレートの各ウェルに細胞の生存および免疫活性化アッセイ細胞懸濁液の500、100〜1,000μLを加える（ 図2A）。 </li><li>静かにプレートを旋回し、5％のCO 2で37℃でO / Nそれらをインキュベートします。百分の50から70の密集度に細胞を増殖させます。 </li><li>トランスフェクションの計画によると、1.5ミリリットルのマイクロチューブ（例、 図2B）に試験RNAおよびそのコントロールの希釈液を準備します。 <ol><li>ウイルス産生アッセイのために、HIV-1発現プラスミド10ngの/μlの希釈を準備し、各チューブに10μlを添加します。次の5μMの試験RNAの希釈液および陰性対照RNを準備A. 25または100 nMの最終濃度のために、対応するチューブに各試験RNAおよび陰性対照希​​釈の2.5または10μLを加えます。 </li><li> C：細胞生存率アッセイのために、試験RNAおよびポジティブコントロールRNA、低分子量ポリIの10 mg / mlで希釈5μMの希釈液を準備します。それぞれ、100 nMまたはそれを200μg/ mlの最終濃度のために、対応するチューブに試験RNAまたは陽性対照RNA希釈液の2μlを添加します。 </li><li>免疫活性化アッセイのために、ステップ1.3.2で作成した試験RNAまたは陽性対照RNA希釈液の20μlのを追加します。それぞれ100 nMもしくは200 / mlの最終濃度、に対応するチューブに。 テストRNA発現プラスミドのために、試験RNAの5μM希釈液の代わりに、10 ngの/μlの希釈液を調製し、ステップ1.3.1のための25および100 ngの最終的な量を与える：注意してください。およびステップ1.3.2のための100 ngの。および1.3.3。 </li></ol></li><li>ウイルスのprodのための各トランスフェクションチューブにDMEMの50、25または75μLを加えますそれぞれuction、細胞の生存および免疫活性化アッセイ。ウイルス産生アッセイのために、次のステップの前にバイオセーフティレベル3（BSL3）研究室への細胞、準備トランスフェクションチューブをもたらします。 </li><li>トランスフェクションチューブにトランスフェクション試薬を順次2μlを添加して、複合体を形成することができるように、15〜20分間インキュベートします。使用される特定のトランスフェクション試薬のための材料の表を参照してください。 </li><li>滴下細胞培養プレートの対応する位置に各マイクロチューブから全トランスフェクション混合物を加えます。ゆっくり旋回し、5％のCO 2で37℃で48時間プレートをインキュベートします。 </li><li>細胞培養プレート中のHIV-1産生（セクション2）、細胞生存率（セクション3）と免疫活性化（セクション4）（ 図2C）を測定します 。 </li></ol> 2.ウイルス産生アッセイ<ol><li>インキュベーターから24ウェル細胞培養プレートを外し、静かにBSL3細胞培養内部のプレートを旋回フード。 HIV-1産生を定量化するために使用される96ウェル平底プレートに対応するウェルに、各ウェルからの上清150μlのを転送します。 注意：一般的なウイルスの定量アッセイは、酵素結合免疫吸着アッセイ（ELISA）20、逆転写ポリメラーゼ連鎖反応（RT-PCR）21によるウイルスRNAを定量することにより、HIV-1キャプシドタンパク質（P24）の発現を測定し、活性を測定することを含みますHIV-1 RT酵素。 2.2 2.5には、HIV-1 RT活性13,15,16を定量化する方法を説明する手順。 </li><li>ウイルスの破壊カクテル15（ 表1）の25μLを含む96ウェルプレートの対応するウェルに上清の5μlを採取し、。室温で5分間混合物をインキュベートし、放射能のワークステーションにプレートを転送します。 注：ステップ2.2。放射能ワークステーションがBSL3実験室で利用できない場合のみ必要です。プレートから除去する必要がない場合BSL3実験室は、2.3に進みます。放射性カクテルを25μlの代わりにウイルス/放射性崩壊カクテル（ 表1）の50μlを添加します。 </li><li>放射性カクテル15（ 表1）を準備し、ウイルス上清と混乱カクテルの各ウェルに25μlを添加します。 2時間37℃で培養します。 </li><li>スポット5ガラス繊維ディエチルアミノエチル（DEAE）フィルターマット紙で対応する四角の上に反応混合物μlのスポットが10分間乾燥させます。どの2つのサンプルが互いに直接ボーダーないように、他のすべての広場に反応混合物を発見。これは、ステップ2.6で分当たりのカウント（cpm）を決定する際にサンプル間のクロスオーバーを回避するのに役立ちます。 </li><li>紙は、95％エタノールで2つの1分間の洗浄、続いて2×生理食塩水クエン酸ナトリウム（SSC）緩衝液（ 表1）を用いて5分間、5倍洗浄します。論文は、サンプルバッグにそれらを乾燥し、密封することを可能にします。 </li><li>クリップサンプルバッグCOカセットにフィルターマット紙をntainingし、マイクロプレートシンチレーションカウンターにカセットを挿入します。実験に使用した[32 P] dTTPのバッチのために提供基準日と32 Pのためのcpmを読むためにカウンタを設定します。プレートマップを使用して、読んで、カウンタを開始するためにどのサンプルを選択します。 </li><li>任意のウイルスの定量法については、隣接する負の制御により、各試験RNAについて得られた値を分割し、試験RNAの各反復のためのHIV-1産生の阻害パーセントを取得するには100で、この値を乗算します。異なる濃度での異なる試験RNAを比較した結果の例を図3に提供されます。 </li></ol> 3.細胞生存率アッセイ<ol><li> / mlのMTT（3- [4.5ジメチル-2-チアゾリル] -2,5-ジフェニル2H-テトラゾリウムブロマイド）ダルベッコのリン酸緩衝生理食塩水で希釈した（インキュベーターから96ウェルプレートを取り外し、5 mgの20μlのを追加各ウェルにDPBS）。 Fプレートをインキュベートまたは37℃で3時間。 </li><li>洗剤（1％NP-40、イソプロパノール中4 mMの塩酸）を各ウェルへと酸性化イソプロパノール150μlを添加して、室温で2時間プレートをインキュベート。 </li><li>マイクロプレート分光光度計で570 nmの吸光度を決定します。 </li><li>隣接するトランスフェクション制御によって各サンプルについて得られた値を割ることにより、各陽性対照および試験RNAの相対MTT代謝を計算します。異なる試験RNAおよび陽性対照を比較した結果の一例を図4に設けられています。 </li></ol> 4.免疫活性化アッセイ<ol><li>インキュベーターから12ウェルプレートを取り外し、培地を吸引除去します。静かDPBSで細胞を2回洗浄し、各ウェルに（プロテアーゼおよびホスファターゼ阻害剤を含む、 表1）冷溶解バッファ22の70μlを添加します。氷上で10分間、プレートをインキュベートします。 </li><li>マイクロチューブに細胞溶解物を移し、高速浸漬することにより、それらを凍結液体窒素中でチューブ。サンプルは解凍と3の合計2以上の凍結融解サイクルの繰り返しを許可します。 </li><li>細胞破片をペレット化し、4℃、15,700×gで15分間、溶解物を遠心分離します。新しいマイクロチューブに上清を移し、クマシーブルー（ブラッドフォード）メソッド23,24を用いてタンパク質濃度を決定します。 </li><li> 10％変性ポリアクリルアミドゲルに各試料からのタンパク質の75μgのを解決し、以前に25,26を説明したように、ニトロセルロース膜に移します。 </li><li>膜に電気泳動転写した後、蒸留水で洗浄し、1分間ポンソーS（ 表1）中で膜をインキュベートすることによってタンパク質バンドを明らかにする。 80と55 kDaので膜をカットするためのガイドとしてバンドおよびタンパク質ラダーを使用してください。 注：カットスルー所定の位置で膜をカットしていないことが容易であるように、ステップでは4.4のサンプルは、34キロダルトンまでの16×18 cm 2のゲル上で実行することができます興味のあるバンド。また、いくつかのゲルは、膜を切断回避するために、同じ試料を用いて実行することができます。 </li><li> 0.05％トゥイーン20（TBST、 表1）を含有するトリス緩衝生理食塩水ポンソーS染色を洗い流します。完全に膜をカバーするために、5％無脂肪乳でTBSTを追加します。 1時間攪拌しながら室温で膜をインキュベートします。 </li><li> 、ADAR1（110と150キロダルトン）、ホスホPKR（62キロダルトン）、およびホスホIRF3（47キロダルトン）に対して千1に希釈した3％ウシ血清アルブミン（BSA）とTBSTで膜をO / N及び抗体をインキュベートそれぞれトップ、膜の中央部および底部片のため。 </li><li>膜をTBSTで5分間、5倍、1時間（5,000 1に希釈）を5％脱脂乳およびペルオキシダーゼ標識ヤギ抗ウサギ二次抗体を用いてTBSTでインキュベート洗います。 </li><li> TBSTで5分間膜5回洗浄し、製造元の指示に従って、フィルム上のバンドを可視化するために電気化学発光（ECL）ソリューションを適用します。 </li><li>フィルム上のタンパク質バンドを可視化した後、抗体剥離液で10分間、膜の中央と下部の部分を洗います。 IRF3（500中1で）総PKRに対する3％BSAを含むTBSTで膜をO / Nおよび抗体をインキュベート（千1時）、それぞれ中間及び底部片のため。繰り返しは、4.8を繰り返します。 4.9。 PKR膜（中央）、抗ウサギ二次抗体の代わりに、ペルオキシダーゼ標識ヤギ抗マウスを用いました。 </li><li> TBSTで5分間膜5倍の底部片を洗浄し（5000で1に希釈）アクチンに対する抗体で1時間、3％BSAを含むTBST中で膜をインキュベートします。繰り返しは、4.8を繰り返します。 4.9。抗ウサギ二次抗体の代わりに、ペルオキシダーゼ標識ヤギ抗マウスを用いました。異なる試験RNAおよびポジティブコントロールを比較した結果の一例を図5に設けられている。使用される特定の抗体のための材料の表を参照してください。 </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal Biochem. 72, 248-254 (1976).</li><li>Stoscheck, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+protein.">Quantitation of protein.</a> Methods Enzymol. 182, 50-68 (1990).</li><li>Battisti, P. L., 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=Additive+activity+between+the+trans-activation+response+RNA-binding+protein,+TRBP2,+and+cyclin+T1+on+HIV+type+1+expression+and+viral+production+in+murine+cells.">Additive activity between the trans-activation response RNA-binding protein, TRBP2, and cyclin T1 on HIV type 1 expression and viral production in murine cells.</a> AIDS Res Hum Retroviruses. 19, 767-778 (2003).</li><li>Bannwarth, S., 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=Cell-specific+regulation+of+TRBP1+promoter+by+NF-Y+transcription+factor+in+lymphocytes+and+astrocytes.">Cell-specific regulation of TRBP1 promoter by NF-Y transcription factor in lymphocytes and astrocytes.</a> J Mol Biol. 355, 898-910 (2006).</li><li>Clerzius, 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=ADAR1+interacts+with+PKR+during+human+immunodeficiency+virus+infection+of+lymphocytes+and+contributes+to+viral+replication.">ADAR1 interacts with PKR during human immunodeficiency virus infection of lymphocytes and contributes to viral replication.</a> J Virol. 83, 10119-10128 (2009).</li><li>Burugu, S., Daher, A., Meurs, E. F., Gatignol, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+translation+and+its+regulation+by+cellular+factors+PKR+and+PACT.">HIV-1 translation and its regulation by cellular factors PKR and PACT.</a> Virus Res. 193, 65-77 (2014).</li><li>ter Brake, O., Konstantinova, P., Ceylan, M., Berkhout, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+of+HIV-1+with+RNA+interference:+a+multiple+shRNA+approach.">Silencing of HIV-1 with RNA interference: a multiple shRNA approach.</a> Mol Ther. 14, 883-892 (2006).</li><li>Naito, 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=Optimal+design+and+validation+of+antiviral+siRNA+for+targeting+HIV-1.">Optimal design and validation of antiviral siRNA for targeting HIV-1.</a> Retrovirology. 4, 80 (2007).</li><li>Mandal, D., Feng, Z., Stoltzfus, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excessive+RNA+splicing+and+inhibition+of+HIV-1+replication+induced+by+modified+U1+small+nuclear+RNAs.">Excessive RNA splicing and inhibition of HIV-1 replication induced by modified U1 small nuclear RNAs.</a> J Virol. 84, 12790-12800 (2010).</li><li>Chang, Z., 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=Enhanced+expression+and+HIV-1+inhibition+of+chimeric+tRNA(Lys3)-ribozymes+under+dual+U6+snRNA+and+tRNA+promoters.">Enhanced expression and HIV-1 inhibition of chimeric tRNA(Lys3)-ribozymes under dual U6 snRNA and tRNA promoters.</a> Mol Ther. 6, 481-489 (2002).</li><li>Chang, L. J., Liu, X., He, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+siRNAs+targeting+multiple+highly+conserved+RNA+sequences+of+human+immunodeficiency+virus+type+1.">Lentiviral siRNAs targeting multiple highly conserved RNA sequences of human immunodeficiency virus type 1.</a> Gene Ther. 12, 1133-1144 (2005).</li><li>Daniels, S. 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=HIV-1+RRE+RNA+acts+as+an+RNA+silencing+suppressor+by+competing+with+TRBP-bound+siRNAs.">HIV-1 RRE RNA acts as an RNA silencing suppressor by competing with TRBP-bound siRNAs.</a> RNA Biol. 12, 123-135 (2015).</li><li>Castanotto, 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=Combinatorial+delivery+of+small+interfering+RNAs+reduces+RNAi+efficacy+by+selective+incorporation+into+RISC.">Combinatorial delivery of small interfering RNAs reduces RNAi efficacy by selective incorporation into RISC.</a> Nucleic Acids Res. 35, 5154-5164 (2007).</li><li>Vickers, T. A., Sabripour, M., Crooke, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=U1+adaptors+result+in+reduction+of+multiple+pre-mRNA+species+principally+by+sequestering+U1snRNP.">U1 adaptors result in reduction of multiple pre-mRNA species principally by sequestering U1snRNP.</a> Nucleic Acids Res. 39, e71 (2011).</li><li>Tai, C. J., Li, C. L., Tai, C. J., Wang, C. K., Lin, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Viral+Entry+Assays+for+the+Identification+and+Evaluation+of+Antiviral+Compounds.">Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds.</a> J Vis Exp. , (2015).</li><li>Riss, T. L., et al., Sittampalam, G. S., 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=.">.</a> Assay Guidance Manual. , (2004).</li><li>Reynolds, 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=Induction+of+the+interferon+response+by+siRNA+is+cell+type-+and+duplex+length-dependent.">Induction of the interferon response by siRNA is cell type- and duplex length-dependent.</a> Rna. 12, 988-993 (2006).</li><li>Whitehead, K. A., Dahlman, J. E., Langer, R. S., Anderson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+or+stimulation?+siRNA+delivery+and+the+immune+system.">Silencing or stimulation? siRNA delivery and the immune system.</a> Annu Rev Chem Biomol Eng. 2, 77-96 (2011).</li></ol>

The authors have nothing to disclose.

HIV-1産生アッセイを説明11,31標的部位をHEK293T細胞（ 図2）を使用して実施し、効果的なリボザイム13、shRNAを10,29のためのHIV-1 RNAをスクリーニングするために使用されるアッセイと類似して、siRNAの30、及びU1i RNAました。 HIV-1産生を定量するために異なる方法を使用して、ほとんどの研究は、候補RNAとHIV-1発現プラスミドのコトランスフェクションし...

現在のHIV-1治療の限界は、それらが慢性疾患の進行を予防するために投与しなければならないことです。 HIV-1抵抗性Tリンパ球または造血幹細胞の移植、薬物療法1,2の非存在下でHIV-1複製の長期制御を提供する可能性があり、また、HIV-1の硬化を達成するのに有効なアプローチであり得ます3。 HIV-1複製に耐性細胞をレンダリングするための一つの方法は、自己移植4中に感染した個体の細胞内への抗HIV-1のRNAまたはペプチドをコードする1つ以上の遺伝子を挿入することです。いくつかの候補の抗HIV-1遺伝子は、任意の単一遺伝子をHIV-1抵抗性の発達を防止するために、2つの5または三6の組み合わせでいくつかの入力の臨床試験を用いて設計されています。 抗HIV-1 RNAは、免疫応答を誘発するそれらの低電位による組み合わせ遺伝子治療のために、彼らため上位候補の一つであります非常に短い遺伝子配列から転写されます。いくつかの抗HIV-1 RNAは、ウイルスの侵入及び統合を標的とするように設計されています。しかし、ほとんどの抗HIV-1 RNAは、ウイルスのライフサイクルの後の積分ステップ（ 図1）を標的とします。統合後の阻害剤は、このようなリボザイム7、shRNAは8とU1i RNAを9として、HIV-1調節タンパク質Tatのまたは改訂1、およびアンチセンスベースのRNAを標的とするHIV-1 RNAに異なる部位を標的、デコイRNAを含みます。抗HIV-1 RNAの有効性を比較するために使用されている方法は、10の候補のRNAをコードする遺伝子で形質導入した細胞におけるウイルス複製を監視し、一過候補RNAを発現するプラスミドでトランスフェクトした細胞におけるウイルス産生を測定し、HIV-1発現プラスミドを含みます-13。我々は以前に新しいリボザイム標的部位13-15ためのHIV-1 RNAをスクリーニングするために、HIV-1産生アッセイを使用しています。これらの方法は、以降、RNAのフォーマットを最適化するために洗練されています干渉分子は、shRNAのようプラスミドDNAから発現または合成siRNA 16として配信します。このアッセイは、ヒト胚性腎臓（HEK）293T細胞から成熟したウイルスの産生を測定し、HIV-1複製サイクルの後の積分ステップ（ 図1）を標的とする阻害剤の効果を比較するために使用することができます。統合前のステップを標的とする阻害剤は、このようなTZM-BL細胞感染性アッセイ17などの代替アッセイは、抗ウイルス効果を評価するために必要とされます。 診療所での抗HIV-1 RNAの送達のための主要な安全上の懸念は、人間のRNAまたはタンパク質、および先天性免疫センサーの活性化に対する潜在的なオフターゲット効果が含まれます。抗HIV-1 siRNAの毒性を評価するために、我々は、異なる細胞株16における細胞生存率アッセイを使用しています。我々はまた、二本鎖RNA、免疫センサーの活性化を測定し、RNA活性化プロテインキナーゼR（PKR）及び受容体3（TLR3）Toll様、並びにEXインターフェロンのPRESSIONは、遺伝子、ADAR1のP150を刺激しました。これらのアッセイは、抗HIV-1 RNAの有効性は、細胞の生存または免疫センサーの活性化の間接的な影響によるものではないことを確認するために使用することができます。彼らはまた、さらなる開発の潜在的な毒性を有する候補RNAを除くのに有用です。 次のプロトコルでは、新しい治療のRNAを識別し、既存のフォーマットを最適化するための手順が記載されています。方法は、HIV-1複製のRNAベースの統合後の阻害剤をスクリーニングするために有用であり、そのようなウイルスRNA 18または統合された標的とするように設計されたCRISPR / CASシステムの改訂媒介輸出を標的とする小分子などの他の統合後の阻害剤をスクリーニングするために適合させることができますHIV-1 DNA 19。

<table><tbody><tr><td>DMEM HyClone</td><td>GE Healthcare</td><td>SH30243.01</td><td></td></tr><tr><td>FBS HyClone</td><td>GE Healthcare</td><td>SH30396.03</td><td></td></tr><tr><td>Penicillin/Streptomycin Gibco</td><td>Thermo Fisher</td><td>15140-122</td><td></td></tr><tr><td>Cell culture plates, 96 well, 24 well, 6 well.</td><td>Corning</td><td>353075, 353047, 353043</td><td></td></tr><tr><td>Micro tubes Axygen</td><td>Corning</td><td>311-08-051</td><td></td></tr><tr><td>Low molecular weight Poly I:C</td><td>InvivoGen</td><td>3182-29-6</td><td></td></tr><tr><td>DharmaFECT-1</td><td>Dharmacon</td><td>T-2001-01</td><td>transfection reagent for synthetic RNAs</td></tr><tr><td>TransIT-LT1</td><td>Mirus</td><td>MIR 2300</td><td>transfection reagent for RNA expression plasmids</td></tr><tr><td>Nonidet P40 (NP-40)</td><td>USB</td><td>19628</td><td></td></tr><tr><td>[&lt;sup&gt;32&lt;/sup&gt;P]dTTP</td><td>Perkin Elmer</td><td>BLU505H</td><td></td></tr><tr><td>poly(A) RNA template&amp;nbsp;</td><td>Sigma-Aldrich</td><td>10108626001</td><td></td></tr><tr><td>oligo(dT)&lt;sub&gt;12-18&lt;/sub&gt; DNA primer</td><td>Thermo Fisher</td><td>18418-012</td><td></td></tr><tr><td>DEAE filtermat paper&amp;nbsp;</td><td>Perkin Elmer</td><td>1450-522</td><td></td></tr><tr><td>Microplate scintillation counter</td><td>Perkin Elmer</td><td>1450-024</td><td></td></tr><tr><td>MTT</td><td>Sigma-Aldrich</td><td>M-2128</td><td></td></tr><tr><td>DPBS HyClone</td><td>GE Healthcare</td><td>SH30028.02</td><td></td></tr><tr><td>Microplate spectrophotometer</td><td>Bio-rad</td><td>1706930</td><td></td></tr><tr><td>Lysis buffer tablets</td><td>Roche</td><td>4693159001, 4906837001</td><td>protease and phosphatase inhibitors</td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>5415R</td><td></td></tr><tr><td>Bradford reagent</td><td>Bio-rad</td><td>500-0006</td><td></td></tr><tr><td>Gel running chamber</td><td>Hoefer</td><td>SE600</td><td></td></tr><tr><td>Semi-dry transfer cell</td><td>Bio-rad</td><td>1703940</td><td></td></tr><tr><td>Protein ladder EZ-Run</td><td>Thermo Fisher</td><td>BP3603-500</td><td></td></tr><tr><td>Nitrocellulose membrane</td><td>Bio-rad</td><td>162-0094</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A9647-1006</td><td></td></tr><tr><td>Antibody stripping solution</td><td>Millipore</td><td>2504</td><td></td></tr><tr><td>ECL - Pierce</td><td>Thermo Fisher&amp;nbsp;</td><td>PI32106</td><td></td></tr><tr><td>ADAR1 antibody</td><td></td><td></td><td>from Dr. B.L. Bass</td></tr><tr><td>phospho-T446-PKR antibody</td><td>Abcam</td><td>ab32036</td><td></td></tr><tr><td>phospho-S396-IRF3 antibody</td><td>Cell Signaling</td><td>4947</td><td></td></tr><tr><td>PKR antibody</td><td></td><td></td><td>from Dr. A. Hovanessian</td></tr><tr><td>IRF3 antibody</td><td>Cell Signaling</td><td>11904</td><td></td></tr><tr><td>Actin antibody</td><td>Millipore</td><td>MAB1501</td><td></td></tr><tr><td>Peroxidase-labeled goat anti-rabbit</td><td>KPL</td><td>474-1506</td><td></td></tr><tr><td>Peroxidase-labeled goat anti-mouse</td><td>KPL</td><td>474-1806</td><td></td></tr><tr><td>Ponceau S&amp;nbsp;</td><td>Sigma-Aldrich</td><td>6226-79-5</td><td></td></tr></tbody></table>

evaluation of the efficacy and toxicity of rnas targeting hiv-1 production for use in gene or drug therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

手順の一般的な概略図は、3つの試験RNAおよび図2Bに設けられたコントロールRNAのための例示的トランスフェクション計画を図2に示されています。ウイルスの産生および細胞生存率アッセイのために、各試験構築物のための読み出しは、陰性対照に正規化されます。各試験RNAは、その隣接する負の対照に対して標準化されるように複製が、セ?...

Watch this Scientific Journal Video about Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy at JoVE.com

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

遺伝子または薬物療法で使用するためのHIV-1産生を標的RNAの有効性および毒性の評価

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

evaluation-efficacy-toxicity-rnas-targeting-hiv-1-production-for-use

Research

JoVE Journal

Immunology and Infection

7.8K ビュー.  Lady Davis Institute for Medical Research. Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones. 

ビデオ: Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Here we describe a protocol for employing high-throughput RNAi screening to uncover host targets that can be manipulated to enhance oncolytic virus therapy, specifically rhabodvirus and vaccinia virus therapy, but it can be readily adapted to other oncolytic virus platforms or for discovering host genes that modulate virus replication generally.

腫瘍溶解性ウイルス治療を調節するホストの要因を識別するために全ゲノム RNAi スクリーニング

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus ...

がん研究

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

siRNA導入のための細胞型に特異的な抗HIV gp120のアプタマーの開発

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

免疫・感染学

DNA Vector-based RNA Interference to Study Gene Function in Cancer

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing1, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible2,3. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. 
We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. 
Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer4,5, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.

RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo.

癌における遺伝子機能を研究するDNAベクターベースのRNA干渉

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA ...

医学

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNAウイルスの広域スペクトルの化学抑制剤のためのハイスループットスクリーニング

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

HIV逆転写酵素及びインテグラーゼ阻害剤の迅速なスクリーニング

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter &beta;1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

RNAiのエフェクターの細菌配達：Transkingdom RNAiの

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

生物学

MicroRNA-based Regulation of Picornavirus Tropism

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and understanding the biological properties of viruses. There are several mechanisms for regulating viral tropism, however they tend to be virus class specific and many result in virus attenuation. Additionally, many viruses, including picornaviruses, exhibit size constraints that do not allow for incorporation of large amounts of foreign genetic material required for some targeting methods. MicroRNAs are short, non-coding RNAs that regulate gene expression in eukaryotic cells by binding complementary target sequences in messenger RNAs, preventing their translation or accelerating their degradation. Different cells exhibit distinct microRNA signatures and many microRNAs serve as biomarkers. These differential expression patterns can be exploited for restricting gene expression in cells that express specific microRNAs while maintaining expression in cells that do not. In regards to regulating viral tropism, sequences complementary to specific microRNAs are incorporated into the viral genome, generally in the 3' non-coding regions, targeting them for destruction in the presence of the cognate microRNAs thus preventing viral gene expression and/or replication. MicroRNA-targeting is a technique that theoretically can be applied to all viral vectors without altering the potency of the virus in the absence of the corresponding microRNAs. Here we describe experimental methods associated with generating a microRNA-targeted picornavirus and evaluating the efficacy and specificity of that targeting in vitro. This protocol is designed for a rapidly replicating virus with a lytic replication cycle, however, modification of the time points analyzed and the specific virus titration readouts used will aid in the adaptation of this protocol to many different viruses.

We describe here a method for regulating picornavirus tropism by incorporating sequences complementary to specific microRNAs into the viral genome. This protocol can be adapted to all different classes of viruses with modifications based upon the length and nature of their life cycle.

ピコルナウイルス向性のマイクロRNAベースの規制

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and ...

High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune system and is not targeted by the current antiretroviral therapy (cART). Transcription and splicing have been shown to reinforce HIV-1 latency in resting CD4+ T cells. Reversal of latency by the use of latency reversal agents (LRAs) in the &#34;shock and kill&#34; approach has been studied extensively in an attempt to purge this reservoir but has thus far not shown any success in clinical trials due to the lack of development of adequate small molecules that can efficiently perturb this reservoir. The protocol presented here provides a method for reliably and efficiently assessing latency reversal agents (LRAs) on HIV transcription and splicing. This approach is based on the use of an LTR-driven dual color reporter that can simultaneously measure the effect of an LRA on transcription and splicing by flow cytometry. The protocol described here is adequate for adherent cells as well as the cells in suspension. It is useful for testing a large number of drugs in a high throughput system. The method is technically simple to implement and cost-effective. In addition, the use of flow cytometry allows the assessment of cell viability and thus drug toxicity at the same time.

A high throughput protocol for functional assessment of HIV efficient reactivation and clearance of latent proviruses is described and applied by testing the impact of interventions on HIV transcription and splicing. Representative results of the effect of latency reversing agents on LTR-driven transcription and splicing are provided.

In Vitro 逆に HIV 転写、スプライシングのエージェントの待機時間の高スループット

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

蛍光によるウイルスRNAの検出<em&gt;その場で</em&gt;ハイブリダイゼーション（FISH）

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Oral Combinational Antiretroviral Treatment in HIV-1 Infected Humanized Mice

The human immunodeficiency virus (HIV-1) pandemic continues to spread unabated worldwide, and currently, there is no vaccine available against HIV. Although combinational antiretroviral therapy (cART) has been successful in suppressing viral replication, it cannot completely eradicate the reservoir from HIV-infected individuals. A safe and effective cure strategy for HIV infection will require multipronged methods, and therefore the advancements of animal models for HIV-1 infection are pivotal for the development of HIV cure research. Humanized mice recapitulate key features of HIV-1 infection. The humanized mouse model can be infected by HIV-1 and viral replication can be controlled with cART regimens. Moreover, cART interruption results in a prompt viral rebound in humanized mice. However, administration of cART to the animal can be ineffective, difficult, or toxic, and many clinically relevant cART regimens are unable to be optimally utilized. Along with being potentially unsafe for researchers, administration of cART by a commonly used intensive daily injection procedure induces stress by physical restraint of the animal. The novel oral cART method to treat HIV-1 infected humanized mice described in this article resulted in suppression of viremia below the detection level, increased rate of CD4+ restoration, and improved overall health in HIV-1 infected humanized mice.

This protocol describes a novel method to deliver oral combinational antiretroviral drugs that successfully suppress HIV-1 RNA replication in humanized mice.

HIV-1感染ヒト化マウスにおける経口併用抗レトロウイルス治療

The human immunodeficiency virus (HIV-1) pandemic continues to spread unabated worldwide, and currently, there is no vaccine available against HIV. ...