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.).

1. Cells and Transfections
<ol>
	<li>Culture HEK 293T cells in Dulbecco&#39;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 &#181;l 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).</li>
	<li>Gently swirl the plates and incubate them O/N at 37 &#176;C with 5% CO2. Grow cells to 50-70% confluency.</li>
	<li>According to a transfection plan, prepare dilutions of test RNAs and their controls in 1.5 ml micro tubes (Example, Figure 2B).
	<ol>
		<li>For the viral production assay, prepare a 10 ng/&#181;l dilution of an HIV-1 expression plasmid and add 10 &#181;l to each tube. Next prepare 5 &#181;M dilutions of test RNAs and a negative control RNA. Add 2.5 or 10 &#181;l of each test RNA and negative control dilution to the corresponding tubes for 25 or 100 nM final concentrations.</li>
		<li>For the cell viability assay, prepare 5 &#181;M dilutions of test RNAs and a 10 mg/ml dilution of the positive control RNA, low molecular weight Poly I:C. Add 2 &#181;l of test RNA or positive control RNA dilutions to the corresponding tubes for 100 nM or 200 &#181;g/ml final concentrations, respectively.</li>
		<li>For the immune activation assay, add 20 &#181;l of test RNA or positive control RNA dilutions prepared in step 1.3.2. to the corresponding tubes for 100 nM or 200 &#181;g/ml final concentrations, respectively. 
		Note: For testing RNA expression plasmids, prepare 10 ng/&#181;l dilutions in place of the 5 &#181;M 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.</li>
	</ol>
	</li>
	<li>Add 50, 25 or 75 &#181;l 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.</li>
	<li>Add 2 &#181;l 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.</li>
	<li>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 &#176;C with 5% CO2.</li>
	<li>Measure HIV-1 production (section 2), cell viability (section 3) and immune activation (section 4) in the cell culture plates (Figure 2C).</li>
</ol>
2. Viral Production Assay
<ol>
	<li>Remove the 24-well cell culture plates from the incubator and gently swirl the plates inside the BSL3 cell culture hood. Transfer 150 &#181;l 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 activity13,15,16.</li>
	<li>Transfer 5 &#181;l of supernatant to corresponding wells in a 96-well plate, containing 25 &#181;l 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 &#181;l of radioactive/viral disruption cocktail (Table 1) in place of the 25 &#181;l of radioactive cocktail.</li>
	<li>Prepare a radioactive cocktail15 (Table 1), and add 25 &#181;l to each well of viral supernatant and disruption cocktail. Incubate the plates at 37 &#176;C for 2 hr.</li>
	<li>Spot 5 &#181;l 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Cell Viability Assay
<ol>
	<li>Remove the 96-well plates from the incubator and add 20 &#181;l of 5 mg/ml MTT (3-[4.5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) diluted in Dulbecco&#39;s phosphate buffered saline (DPBS) to each well. Incubate the plates for 3 hr at 37 &#176;C.</li>
	<li>Add 150 &#181;l 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.</li>
	<li>Determine the absorbance at 570 nm in a microplate spectrophotometer.</li>
	<li>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.</li>
</ol>
4. Immune Activation Assay
<ol>
	<li>Remove the 12-well plates from the incubator and aspirate the culture media. Gently wash the cells twice with DPBS and add 70 &#181;l of cold lysis buffer22 (including protease and phosphatase inhibitors, Table 1) to each well. Incubate the plates for 10 min on ice.</li>
	<li>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.</li>
	<li>Centrifuge the lysates for 15 min at 4 &#176;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) method23,24.</li>
	<li>Resolve 75 &#181;g of protein from each sample in a 10% denaturing polyacrylamide gel and transfer to a nitrocellulose membrane as previously described25,26.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Wash the membranes 5x for 5 min with TBST and apply electrochemiluminescence (ECL) solution to visualize the bands on films according the manufacturer&#39;s instructions.</li>
	<li>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).</li>
	<li>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.</li>
</ol>

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<li>Reynolds, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+the+interferon+response+by+siRNA+is+cell+type-+and+duplex+length-dependent%5BTitle%5D)+AND+%22Rna%22%5BJournal%5D)">Induction of the interferon response by siRNA is cell type- and duplex length-dependent.</a> Rna. 12, 988-993 (2006).</li>
<li>Whitehead, K. A., Dahlman, J. E., Langer, R. S., Anderson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Silencing+or+stimulation%3F+siRNA+delivery+and+the+immune+system%5BTitle%5D)+AND+%22Annu+Rev+Chem+Biomol+Eng%22%5BJournal%5D)">Silencing or stimulation? siRNA delivery and the immune system.</a> Annu Rev Chem Biomol Eng. 2, 77-96 (2011).</li>
</ol>

The authors have nothing to disclose.

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...

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&#39;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 plasmid10-13. We have previously used an HIV-1 production assay to screen HIV-1 RNA for new ribozyme target sites13-15. 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.

<table><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>[&lt;sup&gt;32&lt;/sup&gt;P]dTTP</td><td>Perkin Elmer</td><td>BLU505H</td><td></td></tr><tr><td>poly(A) RNA template&amp;nbsp;</td><td>Sigma-Aldrich</td><td>10108626001</td><td></td></tr><tr><td>oligo(dT)&lt;sub&gt;12-18&lt;/sub&gt; DNA primer</td><td>Thermo Fisher</td><td>18418-012</td><td></td></tr><tr><td>DEAE filtermat paper&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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.

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 ...

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.

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 ...

evaluation-efficacy-toxicity-rnas-targeting-hiv-1-production-for-use

Research

JoVE Journal

Immunology and Infection

7.8K Views.  Lady Davis Institute for Medical Research. 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. 

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

Methods Development for Blood Borne Macrophage Carriage of Nanoformulated Antiretroviral Drugs

Nanoformulated drugs can improve pharmacodynamics and bioavailability while serving also to reduce drug toxicities for antiretroviral (ART) medicines. To this end, our laboratory has applied the principles of nanomedicine to simplify ART regimens and as such reduce toxicities while improving compliance and drug pharmacokinetics. Simple and reliable methods for manufacturing nanoformulated ART (nanoART) are shown. Particles of pure drug are encapsulated by a thin layer of surfactant lipid coating and produced by fractionating larger drug crystals into smaller ones by either wet milling or high-pressure homogenization. In an alternative method free drug is suspended in a droplet of a polymer. Herein, drug is dissolved within a polymer then agitated by ultrasonication until individual nanosized droplets are formed. Dynamic light scattering and microscopic examination characterize the physical properties of the particles (particle size, charge and shape). Their biologic properties (cell uptake and retention, cytotoxicity and antiretroviral efficacy) are determined with human monocyte-derived macrophages (MDM). MDM are derived from human peripheral blood monocytes isolated from leukopacks using centrifugal elutriation for purification. Such blood-borne macrophages may be used as cellular transporters for nanoART distribution to human immunodeficiency virus (HIV) infected organs. We posit that the repackaging of clinically available antiretroviral medications into nanoparticles for HIV-1 treatments may improve compliance and positively affect disease outcomes.

Nanoparticles of indinavir, ritonavir, efavirenz and atazanavir were manufactured using wet milling, homogenization and ultrasonication. These nanoformulations, collectively termed nanoformulated antiretroviral therapy (nanoART), assessed macrophage-based drug delivery. Monocyte-derived macrophage nanoART uptake, retention and sustained release were determined. These preliminary studies suggest the potential of nanoART for clinical use.

Nanoformulated drugs can improve pharmacodynamics and bioavailability while serving also to reduce drug toxicities for antiretroviral (ART) medicines. To ...

Bioengineering

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Here we describe a protocol for employing high-throughput RNAi screening to uncover host targets that can be manipulated to enhance oncolytic virus therapy, specifically rhabodvirus and vaccinia virus therapy, but it can be readily adapted to other oncolytic virus platforms or for discovering host genes that modulate virus replication generally.

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus ...

Cancer Research

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

DNA Vector-based RNA Interference to Study Gene Function in Cancer

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing1, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible2,3. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. 
We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. 
Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer4,5, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.

RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo.

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA ...

Medicine

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

Intrasplenic AAV8-shRNA Injection for Efficient and Safe Liver Gene Knockdown

Efficient Gene Knockdown in the Liver via Intrasplenic Injection of Adeno-Associated Virus Serotype 8 (AAV8)-Delivered Small Hairpin RNA

The liver is a major organ that performs essential metabolic functions. Developing an efficient and safe method to knock down gene expression in the liver provides an important tool for determining gene function in liver pathophysiology. In this study, we describe a method for intrasplenic injection of adeno-associated virus serotype 8 (AAV8) engineered to express a small hairpin RNA (shRNA) against a target gene of interest, nucleostemin (NS). Intrasplenic injection of AAV8 expressing an NS-targeting shRNA (AAV8-shNS1) achieved the same knockdown efficiency of NS in the liver as did portal vein injection, compared to the injection of AAV8 expressing a scrambled sequence shRNA (AAV8-shScr). Furthermore, injection of the AAV8-shRNA virus triggered minimal inflammatory reactions in the liver parenchyma. Most importantly, this intrasplenic injection protocol was not technically demanding and caused minimal bleeding at the injection site, which is the leading cause of perioperative and postoperative mortality when performing portal vein injection. This study reports an improved and relatively safe method to achieve efficient gene knockdown in the liver.

This protocol describes how intrasplenic injection of AAV8-delivered small hairpin RNA achieves the same gene knockdown efficiency in the liver as portal vein injection, representing a simpler procedure with much lower perioperative and postoperative mortality and complications.

The liver is a major organ that performs essential metabolic functions. Developing an efficient and safe method to knock down gene expression in the liver ...

Quantitative Structure-Activity Relationship, Activity Prediction, and Molecular Dynamics of Non-nucleotide Reverse Transcriptase Inhibitors

The increasing incidence of HIV-1 drug resistance presents a challenge to the effectiveness of combination antiretroviral therapy, particularly in Southern Africa. The development of resistance to non-nucleotide reverse transcriptase inhibitors (NNRTIs) threatens the long-term success of antiretroviral therapy. In 2019, antimicrobial resistance directly accounted for an estimated 1.27 million deaths globally. This study employed an in-silico approach to investigate NNTRI drugs and their derivatives. Techniques used included density functional theory calculations, molecular docking, enumeration, quantitative structure-activity relationship (QSAR) analysis, molecular dynamics simulation (MDS), and molecular mechanics with generalized Born and surface area methods. The analysis focused on various pyrimidine derivatives and six NNRTI drugs, examining their interactions with the HIV-1 protein (PDB code 1HQU).
A QSAR model was developed to predict the biological activity of the six NNRTIs under investigation. Using 94 pyrimidine derivatives, the QSAR model achieved an R2 OF 0.822 and a Q2 of 0.815, indicating a high level of predictive accuracy.
MDS were conducted to assess the stability of various ligands and their newly developed alternatives, ensuring they remained bound to the protein&#39;s active site over a 200-nanosecond simulation timeframe. Etravirine exhibited root mean square deviation (RMSD) fluctuations of approximately 4.5 &#197;, while its enumerated derivatives showed RMSD fluctuations of 3.5 &#197;. Through molecular docking, MDS, and free energy computations, enumerated Etravirine demonstrated the best performance, with an activity value of 7.373 and a docking score of -10.517 kcal/mol. Furthermore, the calculated free energy of binding for enumerated Etravirine was -89.684 kcal/mol, outperforming other ligands under investigation. The significant improvement suggests that the modified Etravirine holds promising potential as a novel agent in antiretroviral therapy.
The obtained lower RMSD value, the enhanced amino acid interactions, and the highest binding free energy indicate that enumerated Etravirine could serve as a viable alternative for HIV/AIDS treatment.

This study used in-silico strategies to identify Enumerated Etravirine as a promising therapeutic agent for HIV. Our findings on molecular interactions and dynamics support the rational design of novel NNRTIs as possible HIV treatment alternatives.

The increasing incidence of HIV-1 drug resistance presents a challenge to the effectiveness of combination antiretroviral therapy, particularly in ...

Chemistry

High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune system and is not targeted by the current antiretroviral therapy (cART). Transcription and splicing have been shown to reinforce HIV-1 latency in resting CD4+ T cells. Reversal of latency by the use of latency reversal agents (LRAs) in the &#34;shock and kill&#34; approach has been studied extensively in an attempt to purge this reservoir but has thus far not shown any success in clinical trials due to the lack of development of adequate small molecules that can efficiently perturb this reservoir. The protocol presented here provides a method for reliably and efficiently assessing latency reversal agents (LRAs) on HIV transcription and splicing. This approach is based on the use of an LTR-driven dual color reporter that can simultaneously measure the effect of an LRA on transcription and splicing by flow cytometry. The protocol described here is adequate for adherent cells as well as the cells in suspension. It is useful for testing a large number of drugs in a high throughput system. The method is technically simple to implement and cost-effective. In addition, the use of flow cytometry allows the assessment of cell viability and thus drug toxicity at the same time.

A high throughput protocol for functional assessment of HIV efficient reactivation and clearance of latent proviruses is described and applied by testing the impact of interventions on HIV transcription and splicing. Representative results of the effect of latency reversing agents on LTR-driven transcription and splicing are provided.

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...