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
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?...

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J., 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=96+shRNAs+designed+for+maximal+coverage+of+HIV-1+variants.">96 shRNAs designed for maximal coverage of HIV-1 variants.</a> Retrovirology. 6, 55 (2009).</li><li>Sajic, R., 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=Use+of+modified+U1+snRNAs+to+inhibit+HIV-1+replication.">Use of modified U1 snRNAs to inhibit HIV-1 replication.</a> Nucleic Acids Res. 35, 247-255 (2007).</li><li>Low, J. T., 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=SHAPE-directed+discovery+of+potent+shRNA+inhibitors+of+HIV-1.">SHAPE-directed discovery of potent shRNA inhibitors of HIV-1.</a> Mol Ther. 20, 820-828 (2012).</li><li>Scarborough, R. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>[32P]dTTP</td>
			<td>Perkin Elmer</td>
			<td>BLU505H</td>
			<td></td>
		</tr>
		<tr>
			<td>poly(A) RNA template&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>10108626001</td>
			<td></td>
		</tr>
		<tr>
			<td>oligo(dT)12-18 DNA primer</td>
			<td>Thermo Fisher</td>
			<td>18418-012</td>
			<td></td>
		</tr>
		<tr>
			<td>DEAE filtermat paper&#160;</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&#160;</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&#160;</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 ...

Research

JoVE Journal

Immunology and Infection

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

V3の人口ベースのシーケンシングを用いたHIV - 1トロピズムの遺伝子型推論

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

ナチュラルキラー細胞傷害性アッセイにおけるHIV - 1感染プライマリCD4 + T細胞を標的細胞としての準備と使用

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

同種造血幹細胞移植後のウイルス感染を予防/治療するMultivirus -特異的T細胞の生成

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

標準的な臨床アッセイによる検出限界以下のウイルス量とHIV感染個体におけるHIV - 1 RNAを増幅して定量化

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

In vitro Uncoating of HIV-1 Cores

In vitro Uncoating of HIV-1 Cores

HIV - 1コアのin vitroでの脱殻で

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

合成脂質二重膜上でHIV-1エンベロープ誘発性ウイルス学的シナプスとシグナリングのイメージング

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

細胞ベースのELISAアッセイを使用して、HIV-1エンベロープ糖タンパク質三量体の構造評価

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

ハイスループット定量的リアルタイムRT-PCRアッセイのタイプIおよびIIIインターフェロンサブタイプの発現プロファイルを決定するための

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

HIV-1陽性個体の血漿からエキソソームの単離

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

の測定<em&gt;インビトロ</em&gt; HIV-1プレインテグレーション複合体の統合活動

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...