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Immunology and Infection

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

Published: September 5th, 2016



1Virus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research, 2Department of Microbiology & Immunology, McGill University, 3Department of Medicine, McGill University

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.

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.

A limitation of current HIV-1 treatments is that they must be chronically administered to prevent disease progression. Transplant of HIV-1 resistant T lymphocyte, or hematopoietic stem cells, has the potential to provide long term control of HIV-1 replication in the absence of drug therapy1,2 and may also be an effective approach to attain an HIV-1 cure3. One way to render cells resistant to HIV-1 replication is to insert one or more genes coding for anti-HIV-1 RNAs or peptides into an infected individual's cells during an autologous transplant4. Several candidate anti-HIV-1 genes have been designed with some entering clinical tria....

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1. Cells and Transfections

  1. Culture HEK 293T cells in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Prepare a 2 x 105 cells/ml suspension in the cell culture medium. Add 500, 100 and 1,000 µl of the cell suspension to each well of 24-well, 96-well and 12-well plates, for viral production, cell viability and immune activation assays, respectively (Figure 2A).
  2. Gently swirl the plates and incuba.......

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A general schematic of the procedures is shown in Figure 2 with an example transfection plan for three test RNAs and a control RNA provided in Figure 2B. For viral production and cell viability assays, the read-out for each test construct is normalized to a negative control. Replicates are transfected in sets, so that each test RNA is normalized to its adjacent negative control. This is done to avoid inaccurate data related to the time between complexing .......

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The HIV-1 production assay described was performed using HEK293T cells (Figure 2) and is similar to assays used to screen HIV-1 RNA for effective ribozyme13, shRNA10,29, siRNA30, and U1i RNA11,31 target sites. Using different methods to quantify HIV-1 production, most studies have measured viral production 48 hr after co-transfection of an HIV-1 expression plasmid with candidate RNAs. Following the production of HIV-1, immature virions undergo proteolytic cleav.......

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The work presented here was supported by the Canadian Institutes of Health Research (CIHR) (grants DCB-120266, PPP-133377 and HBF-348967 to A.G.).


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Name Company Catalog Number Comments
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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