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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 trials in combinations of two⁵ or three⁶, to prevent the development of HIV-1 resistance to any single gene.

Anti-HIV-1 RNAs are among the top candidates for combination gene therapy due to their low potential to elicit immune responses and because they are transcribed from very short gene sequences. Some anti-HIV-1 RNAs have been designed to target viral entry and integration. However, most anti-HIV-1 RNAs target post-integration steps in the viral life cycle (Figure 1). Post-integration inhibitors include decoy RNAs, targeting the HIV-1 regulatory proteins Tat or Rev¹, and antisense-based RNAs, targeting different sites in HIV-1 RNA, such as ribozymes⁷, shRNAs⁸ and U1i RNAs⁹. Methods that have been used to compare the efficacy of anti-HIV-1 RNAs include monitoring viral replication in cells transduced with genes coding for candidate RNAs and measuring viral production in cells transiently transfected with plasmids expressing candidate RNAs and an HIV-1 expression plasmid^10-13. We have previously used an HIV-1 production assay to screen HIV-1 RNA for new ribozyme target sites^13-15. These methods have since been refined to optimize the format of an RNA interference molecule expressed from plasmid DNA as an shRNA or delivered as a synthetic siRNA¹⁶. The assay measures the production of mature viruses from human embryonic kidney (HEK) 293T cells, and can be used to compare the effects of inhibitors that target post-integration steps in the HIV-1 replication cycle (Figure 1). For inhibitors that target pre-integration steps, alternative assays such as a TZM-bl cell infectivity assay¹⁷ are needed to evaluate antiviral efficacy.

Major safety concerns for the delivery of anti-HIV-1 RNAs in the clinic include potential off-target effects on human RNAs or proteins, and activation of innate immune sensors. To evaluate the toxicity of anti-HIV-1 siRNAs, we have used a cell viability assay in different cell lines¹⁶. We also measured activation of the double stranded RNA immune sensors, RNA activated protein kinase R (PKR) and Toll like receptor 3 (TLR3), as well as expression of the interferon stimulated gene, ADAR1 p150. These assays can be used to confirm that the efficacy of anti-HIV-1 RNAs is not due to indirect effects on cell viability or immune sensor activation. They are also useful in excluding candidate RNAs with potential toxicities from further development.

In the following protocols, procedures to identify new therapeutic RNAs and optimize the format of existing ones are described. The methods are useful for screening RNA based post-integration inhibitors of HIV-1 replication and could be adapted to screen other post-integration inhibitors, such as small molecules targeting Rev mediated export of viral RNA¹⁸ or CRISPR/Cas systems designed to target integrated HIV-1 DNA¹⁹.

Protokół

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 test RNAs and their controls in 1.5 ml micro tubes (Example, Figure 2B).
   1. For the viral production assay, prepare a 10 ng/µl dilution of an HIV-1 expression plasmid and add 10 µl to each tube. Next prepare 5 µM dilutions of test RNAs and a negative control RNA. Add 2.5 or 10 µl of each test RNA and negative control dilution to the corresponding tubes for 25 or 100 nM final concentrations.
   2. For the cell viability assay, prepare 5 µM dilutions of test RNAs and a 10 mg/ml dilution of the positive control RNA, low molecular weight Poly I:C. Add 2 µl of test RNA or positive control RNA dilutions to the corresponding tubes for 100 nM or 200 µg/ml final concentrations, respectively.
   3. For the immune activation assay, add 20 µl of test RNA or positive control RNA dilutions prepared in step 1.3.2. to the corresponding tubes for 100 nM or 200 µg/ml final concentrations, respectively.
      Note: For testing RNA expression plasmids, prepare 10 ng/µl dilutions in place of the 5 µM dilutions of test RNAs, giving final amounts of 25 and 100 ng for step 1.3.1. and 100 ng for steps 1.3.2. and 1.3.3.
4. Add 50, 25 or 75 µl of DMEM to each transfection tube for viral production, cell viability and immune activation assays, respectively. For viral production assays, bring cells and prepared transfection tubes to a bio-safety level 3 (BSL3) laboratory before the next step.
5. Add 2 µl of the transfection reagent sequentially to the transfection tubes and incubate 15 to 20 min to allow complexes to form. See the Table of Materials for the specific transfection reagent to be used.
6. Add the entire transfection mixture from each micro tube drop-wise to the corresponding positions in the cell culture plates. Gently swirl and incubate the plates for 48 hr at 37 °C with 5% CO₂.
7. Measure HIV-1 production (section 2), cell viability (section 3) and immune activation (section 4) in the cell culture plates (Figure 2C).

2. Viral Production Assay

1. Remove the 24-well cell culture plates from the incubator and gently swirl the plates inside the BSL3 cell culture hood. Transfer 150 µl of supernatant from each well to a corresponding well in a 96-well flat bottom plate, which will be used to quantify HIV-1 production.
   Note: Common viral quantification assays include measuring the expression of the HIV-1 capsid protein (p24) by enzyme-linked immunosorbent assay (ELISA)²⁰, quantifying viral RNA by reverse transcription polymerase chain reaction (RT-PCR)²¹ and measuring the activity of the HIV-1 RT enzyme. Steps 2.2 to 2.5 explain methods to quantify HIV-1 RT activity^13,15,16.
2. Transfer 5 µl of supernatant to corresponding wells in a 96-well plate, containing 25 µl of a viral disruption cocktail¹⁵ (Table 1). Incubate the mixture for 5 min at RT and transfer the plate to a radioactivity workstation.
   Note: Step 2.2. is only necessary if a radioactivity workstation is not available in the BSL3 laboratory. If the plates do not need to be removed from the BSL3 laboratory, proceed to step 2.3. and add 50 µl of radioactive/viral disruption cocktail (Table 1) in place of the 25 µl of radioactive cocktail.
3. Prepare a radioactive cocktail¹⁵ (Table 1), and add 25 µl to each well of viral supernatant and disruption cocktail. Incubate the plates at 37 °C for 2 hr.
4. Spot 5 µl of the reaction mixture onto corresponding squares in a glass fiber Di Ethyl Amino Ethyl (DEAE) filtermat paper and allow the spots to dry for 10 min. Spot the reaction mixture on every other square so that no two samples directly boarder one another. This helps to avoid cross-over between samples when determining counts per minute (cpm) in step 2.6.
5. Wash the papers 5x for 5 min with 2x saline sodium citrate (SSC) buffer (Table 1), followed by two 1 min washes with 95% ethanol. Allow the papers to dry and seal them in sample bags.
6. Clip sample bags containing the filtermat paper into a cassette and insert the cassette into a microplate scintillation counter. Set the counter to read cpm for ³²P with the reference date provided for the batch of [³²P]dTTP used in the experiment. Select which samples to read using the plate map and start the counter.
7. For any viral quantification method, divide the values obtained for each test RNA by the adjacent negative control and multiply this value by 100 to get the percent inhibition of HIV-1 production for each replicate of the test RNAs. An example of results comparing different test RNAs at different concentrations is provided in Figure 3.

3. Cell Viability Assay

1. Remove the 96-well plates from the incubator and add 20 µl of 5 mg/ml MTT (3-[4.5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) diluted in Dulbecco's phosphate buffered saline (DPBS) to each well. Incubate the plates for 3 hr at 37 °C.
2. Add 150 µl of acidified isopropanol with detergent (1% NP-40, 4 mM HCl in isopropanol) to each well and incubate the plates for 2 hr at RT.
3. Determine the absorbance at 570 nm in a microplate spectrophotometer.
4. Calculate the relative MTT metabolism for each positive control and test RNA by dividing the value obtained for each sample by its adjacent transfection control. An example of results comparing different test RNAs and a positive control is provided in Figure 4.

4. Immune Activation Assay

1. Remove the 12-well plates from the incubator and aspirate the culture media. Gently wash the cells twice with DPBS and add 70 µl of cold lysis buffer²² (including protease and phosphatase inhibitors, Table 1) to each well. Incubate the plates for 10 min on ice.
2. Transfer cell lysates to micro tubes and fast freeze them by immersing the tubes in liquid nitrogen. Allow the samples to thaw and repeat for 2 more freeze thaw cycles for a total of 3.
3. Centrifuge the lysates for 15 min at 4 °C, 15,700 x g, to pellet cell debris. Transfer the supernatant to new micro tubes and determine the protein concentration using the Coomassie blue (Bradford) method^23,24.
4. Resolve 75 µg of protein from each sample in a 10% denaturing polyacrylamide gel and transfer to a nitrocellulose membrane as previously described^25,26.
5. Following electrophoresis and transfer to a membrane, reveal protein bands by incubating the membrane in Ponceau S (Table 1) for 1 min followed by washing in double distilled water. Use the bands and protein ladder as a guide to cut the membrane at 80 and 55 kDa.
   Note: In step 4.4 samples can be run on 16 x 18 cm² gels down to 34 kDa, so that it is easy to cut the membrane at the specified positions and not cut through the bands of interest. Alternatively, several gels can be run with the same samples to avoid cutting the membrane.
6. Wash out the Ponceau S staining with Tris-buffered saline containing 0.05% Tween 20 (TBST, Table 1). Add TBST with 5% non-fat milk to completely cover the membranes. Incubate the membranes at RT with agitation for 1 hr.
7. Incubate the membranes O/N in TBST with 3% bovine serum albumin (BSA) and antibodies diluted to 1 in 1,000 against ADAR1 (110 and 150 kDa), phospho-PKR (62 kDa), and phospho-IRF3 (47 kDa), for the top, middle and bottom pieces of the membrane, respectively.
8. Wash the membranes 5x for 5 min with TBST and incubate in TBST with 5% non-fat milk and peroxidase-labeled goat anti-rabbit secondary antibodies (diluted to 1 in 5,000) for 1 hr.
9. Wash the membranes 5x for 5 min with TBST and apply electrochemiluminescence (ECL) solution to visualize the bands on films according the manufacturer's instructions.
10. After visualizing the protein bands on films, wash the middle and bottom pieces of the membrane for 10 min with an antibody stripping solution. Incubate the membranes O/N in TBST with 3% BSA and antibodies against total PKR (at 1 in 500) and IRF3 (at 1 in 1,000), for the middle and bottom pieces, respectively. Repeat steps 4.8. and 4.9. using peroxidase-labeled goat anti-mouse in place of the anti-rabbit secondary antibody for the PKR membrane (middle).
11. Wash the bottom piece of the membrane 5x for 5 min with TBST and incubate the membrane in TBST with 3% BSA for 1 hr with an antibody against actin (diluted to 1 in 5,000). Repeat steps 4.8. and 4.9. using peroxidase-labeled goat anti-mouse in place of the anti-rabbit secondary antibody. An example of results comparing different test RNAs and a positive control is provided in Figure 5. See the Table of Materials for the specific antibodies to be used.

Wyniki

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 ...

Dyskusje

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...

Materiały

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