Summary

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

Published: September 05, 2016
doi:

Summary

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.

Abstract

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.

Introduction

的当前的HIV-1治疗的局限性在于,它们必须长期施用,以防止疾病的进展。 HIV-1的抗T淋巴细胞,或造血干细胞的移植,有可能提供HIV-1复制的长期控制在没有药物治疗1,2的电位,也可以是实现一种HIV-1治疗的有效方法3。渲染到HIV-1复制抗性细胞的一种方法是自体移植4中插入一个或多个基因编码抗-HIV-1的RNA或肽进入受感染个体的细胞。几个候选抗HIV-1基因已被设计有一些进入临床试验在两个5或3个6的组合,以防止HIV-1的抗性的发展的任何单一基因。

抗HIV-1 RNA的顶端候选组合的基因治疗中,由于它们的低电势以引起免疫反应和,因为它们从很短的基因序列转录。一些抗HIV-1的RNA已被设计为靶向病毒进入和整合。然而,大多数的抗HIV-1的RNA靶向病毒生命周期积分后步骤( 图1)。后的整合酶抑制剂包括诱饵的RNA,针对HIV-1调控蛋白达或REV 1,和基于反义RNA的,在HIV-1 RNA针对不同的网站,如核酶7,8的shRNA的U1i和RNA的9。已用于比较的抗HIV-1 RNA的功效的方法包括监测病毒复制与编码基因候选RNA和测定用表达候选RNA的质粒瞬时转染细胞的病毒的生产和HIV-1的表达质粒转染细胞10 -13。我们先前使用过的HIV-1的生产试验,筛选新的核酶靶位点13-15 HIV-1 RNA。这些方法,至今已细化到优化的RNA的格式干扰分子从质粒DNA表达为一个shRNA的或作为合成的siRNA 16传递。该测定测量生产的人胚胎肾(HEK)293T细胞成熟的病毒,并且可以用于比较针对在HIV-1复制周期积分后步骤( 图1)抑制剂的影响。对于面向预集成的步骤抑制剂,需要替代的试验,如TZM-BL细胞感染性测定17来评价抗病毒疗效。

在临床的抗HIV-1 RNA的递送主要的安全问题包括对人类RNA或蛋白质,以及天然免疫传感器的激活潜在的脱靶效应。为了评估抗HIV-1 siRNA的毒性,我们使用在不同的细胞系16的细胞活力测定。我们还测量了双链RNA的免疫传感器的激活,RNA活化蛋白激酶R(PKR)和Toll样受体3(TLR3),以及移干扰素的PRESSION刺激基因,ADAR1 P150。这些测定可用于证实抗HIV-1 RNA的效力不因对细胞活力或免疫传感器活化间接影响。他们也是不包括候选人的RNA与进一步发展的潜在的毒性非常有用。

在下面的协议,过程,以确定新的治疗RNA和优化现有的格式被描述。该方法可用于HIV-1复制的筛选基于RNA后整合抑制剂和可适于筛选其他集成后抑制剂如小分子靶向启介导的病毒RNA 18或CRISPR的出口/设计CAS大全MSDS系统到目标集成HIV-1 DNA 19。

Protocol

1.细胞转染与培养的HEK 293T细胞在Dulbecco改进的Eagle培养基(DMEM)中补充有10%胎牛血清(FBS)和1%青霉素/链霉素。制备在细胞培养基中的2×10 5个细胞/ ml悬浮液。加500,100和1000微升的细胞悬液以每孔24孔,96孔和12孔板,用于病毒生产,细胞生存力和免疫激活测定法,分别为( 图2A)。 轻轻摇动平板,用5%的CO 2孵育他们O / N在37℃。生长细胞50-70%汇合?…

Representative Results

的程序的一般示意性示出在图2中为三个测试RNA和在图2B中提供了一个对照RNA的一例的转染方案。用于病毒生产和细胞生存力测定法,所读出的每个测试构建体是标准化为阴性对照。重复转染在集,使得每个测试RNA被归一化到其相邻的阴性对照。这样做是为了避免与配位和转染之间的时间,这可能会导致如果,例如,所有的阴性对照的是第一转染不…

Discussion

用HEK293T细胞( 图2)进行描述的HIV-1的生产法和类似用于筛选HIV-1RNA有效核酶13,shRNA10,29,siRNA30,和U1i的RNA 11,31靶位点测定。使用不同的方法来量化的HIV-1的生产,大多数研究已经测量的病毒生产48小时与候选的RNA的HIV-1的表达质粒共转染之后。继生产HIV-1的,不成熟的病毒粒子经历的多聚蛋白的蛋白裂解由HIV-1蛋白酶变得成熟的病毒粒子,能够感染…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这里介绍的工作是由卫生研究院(CIHR)加拿大研究院的支持(授予DCB-120266,PPP-133377和HBF-348967到AG)。

Materials

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

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Citazione di questo articolo
Scarborough, R. J., Adams, K. L., Del Corpo, O., Daher, A., Gatignol, A. Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy. J. Vis. Exp. (115), e54486, doi:10.3791/54486 (2016).

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