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

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

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 therapy^1,2 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....

Protocol

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 incubate them O/N at 37 °C with 5% CO₂. Grow cells to 50-70% confluency.
3. According to a transfection plan, prepare dilutions of te....

Results

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 .......

Discussion

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, shRNA^10,29, siRNA³⁰, and U1i RNA^11,31 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.......

Materials

List of materials used in this article

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