Name: evaluation of the efficacy and toxicity of rnas targeting hiv-1 production for use in gene or drug therapy
Uploaded: 2016-09-05T14:00:00
Description: 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

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 incuba...

<ol>
	<li>Hoxie, J. A., June, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+cell+and+gene+therapies+for+HIV.">Novel cell and gene therapies for HIV.</a> Cold Spring Harb Perspect Med. 2, a007179 (2012).</li><li>Bobbin, M. L., Burnett, J. C., Rossi, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+approaches+for+treatment+of+HIV-1+infection.">RNA interference approaches for treatment of HIV-1 infection.</a> Genome Med. 7, 50 (2015).</li><li>Allers, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+the+cure+of+HIV+infection+by+CCR5Delta32/Delta32+stem+cell+transplantation.">Evidence for the cure of HIV infection by CCR5Delta32/Delta32 stem cell transplantation.</a> Blood. 117, 2791-2799 (2011).</li><li>DiGiusto, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+hematopoietic+stem+cell+based+gene+therapy+for+HIV-1+infection:+considerations+for+proof+of+concept+studies+and+translation+to+standard+medical+practice.">Development of hematopoietic stem cell based gene therapy for HIV-1 infection: considerations for proof of concept studies and translation to standard medical practice.</a> Viruses. 5, 2898-2919 (2013).</li><li>Burke, B. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+Cellular+Resistance+to+HIV-1+Infection+In+Vivo+Using+a+Dual+Therapeutic+Lentiviral+Vector.">Engineering Cellular Resistance to HIV-1 Infection In Vivo Using a Dual Therapeutic Lentiviral Vector.</a> Mol Ther Nucleic Acids. 4, e236 (2015).</li><li>DiGiusto, D. 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J., Berkhout, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+durable+treatment+of+HIV-1+infection+using+RNA+interference.">Toward a durable treatment of HIV-1 infection using RNA interference.</a> Prog Mol Biol Transl Sci. 102, 141-163 (2011).</li><li>Blazquez, L., Fortes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=U1+interference+(U1i)+for+Antiviral+Approaches.">U1 interference (U1i) for Antiviral Approaches.</a> Adv Exp Med Biol. 848, 51-69 (2015).</li><li>McIntyre, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=96+shRNAs+designed+for+maximal+coverage+of+HIV-1+variants.">96 shRNAs designed for maximal coverage of HIV-1 variants.</a> Retrovirology. 6, 55 (2009).</li><li>Sajic, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+modified+U1+snRNAs+to+inhibit+HIV-1+replication.">Use of modified U1 snRNAs to inhibit HIV-1 replication.</a> Nucleic Acids Res. 35, 247-255 (2007).</li><li>Low, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SHAPE-directed+discovery+of+potent+shRNA+inhibitors+of+HIV-1.">SHAPE-directed discovery of potent shRNA inhibitors of HIV-1.</a> Mol Ther. 20, 820-828 (2012).</li><li>Scarborough, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Conserved+Target+Site+in+HIV-1+Gag+RNA+is+Accessible+to+Inhibition+by+Both+an+HDV+Ribozyme+and+a+Short+Hairpin+RNA.">A Conserved Target Site in HIV-1 Gag RNA is Accessible to Inhibition by Both an HDV Ribozyme and a Short Hairpin RNA.</a> Mol Ther Nucleic Acids. 3, e178 (2014).</li><li>Lain&#233;, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+cleavage+of+HIV-1+RNA+by+new+SOFA-HDV+ribozymes+and+their+potential+to+inhibit+viral+replication.">In vitro and in vivo cleavage of HIV-1 RNA by new SOFA-HDV ribozymes and their potential to inhibit viral replication.</a> RNA Biol. 8, 343-353 (2011).</li><li>Scarborough, R. J., L&#233;vesque, M. V., Perreault, J. P., Gatignol, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+Evaluation+of+Clinically+Relevant+SOFA-HDV+Ribozymes+Targeting+HIV+RNA.">Design and Evaluation of Clinically Relevant SOFA-HDV Ribozymes Targeting HIV RNA.</a> Methods Mol Biol. 1103, 31-43 (2014).</li><li>Scarborough, R. J., Adams, K. L., Daher, A., Gatignol, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+Inhibition+of+HIV-1+Production+by+Short+Hairpin+RNAs+and+Small+Interfering+RNAs+Targeting+a+Highly+Conserved+Site+in+HIV-1+Gag+RNA+Is+Optimized+by+Evaluating+Alternative+Length+Formats.">Effective Inhibition of HIV-1 Production by Short Hairpin RNAs and Small Interfering RNAs Targeting a Highly Conserved Site in HIV-1 Gag RNA Is Optimized by Evaluating Alternative Length Formats.</a> Antimicrob Agents Chemother. 59, 5297-5305 (2015).</li><li>Sarzotti-Kelsoe, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+and+validation+of+the+TZM-bl+assay+for+standardized+assessments+of+neutralizing+antibodies+against+HIV-1.">Optimization and validation of the TZM-bl assay for standardized assessments of neutralizing antibodies against HIV-1.</a> J Immunol Methods. 409, 131-146 (2014).</li><li>Campos, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+lasting+control+of+viral+rebound+with+a+new+drug+ABX464+targeting+Rev+-+mediated+viral+RNA+biogenesis.">Long lasting control of viral rebound with a new drug ABX464 targeting Rev - mediated viral RNA biogenesis.</a> Retrovirology. 12, 1-15 (2015).</li><li>Zhu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CRISPR/Cas9+system+inactivates+latent+HIV-1+proviral+DNA.">The CRISPR/Cas9 system inactivates latent HIV-1 proviral DNA.</a> Retrovirology. 12, 22 (2015).</li><li>Bounou, S., Leclerc, J. E., Tremblay, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+host+ICAM-1+in+laboratory+and+clinical+strains+of+human+immunodeficiency+virus+type+1+increases+virus+infectivity+and+CD4(+)-T-cell+depletion+in+human+lymphoid+tissue,+a+major+site+of+replication+in+vivo.">Presence of host ICAM-1 in laboratory and clinical strains of human immunodeficiency virus type 1 increases virus infectivity and CD4(+)-T-cell depletion in human lymphoid tissue, a major site of replication in vivo.</a> J Virol. 76, 1004-1014 (2002).</li><li>Shan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+PCR+assay+for+quantification+of+HIV-1+RNA.">A novel PCR assay for quantification of HIV-1 RNA.</a> J Virol. 87, 6521-6525 (2013).</li><li>Clerzius, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PKR+activator,+PACT,+becomes+a+PKR+inhibitor+during+HIV-1+replication.">The PKR activator, PACT, becomes a PKR inhibitor during HIV-1 replication.</a> Retrovirology. 10, 96 (2013).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal Biochem. 72, 248-254 (1976).</li><li>Stoscheck, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+protein.">Quantitation of protein.</a> Methods Enzymol. 182, 50-68 (1990).</li><li>Battisti, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Additive+activity+between+the+trans-activation+response+RNA-binding+protein,+TRBP2,+and+cyclin+T1+on+HIV+type+1+expression+and+viral+production+in+murine+cells.">Additive activity between the trans-activation response RNA-binding protein, TRBP2, and cyclin T1 on HIV type 1 expression and viral production in murine cells.</a> AIDS Res Hum Retroviruses. 19, 767-778 (2003).</li><li>Bannwarth, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-specific+regulation+of+TRBP1+promoter+by+NF-Y+transcription+factor+in+lymphocytes+and+astrocytes.">Cell-specific regulation of TRBP1 promoter by NF-Y transcription factor in lymphocytes and astrocytes.</a> J Mol Biol. 355, 898-910 (2006).</li><li>Clerzius, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ADAR1+interacts+with+PKR+during+human+immunodeficiency+virus+infection+of+lymphocytes+and+contributes+to+viral+replication.">ADAR1 interacts with PKR during human immunodeficiency virus infection of lymphocytes and contributes to viral replication.</a> J Virol. 83, 10119-10128 (2009).</li><li>Burugu, S., Daher, A., Meurs, E. F., Gatignol, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+translation+and+its+regulation+by+cellular+factors+PKR+and+PACT.">HIV-1 translation and its regulation by cellular factors PKR and PACT.</a> Virus Res. 193, 65-77 (2014).</li><li>ter Brake, O., Konstantinova, P., Ceylan, M., Berkhout, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+of+HIV-1+with+RNA+interference:+a+multiple+shRNA+approach.">Silencing of HIV-1 with RNA interference: a multiple shRNA approach.</a> Mol Ther. 14, 883-892 (2006).</li><li>Naito, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+design+and+validation+of+antiviral+siRNA+for+targeting+HIV-1.">Optimal design and validation of antiviral siRNA for targeting HIV-1.</a> Retrovirology. 4, 80 (2007).</li><li>Mandal, D., Feng, Z., Stoltzfus, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excessive+RNA+splicing+and+inhibition+of+HIV-1+replication+induced+by+modified+U1+small+nuclear+RNAs.">Excessive RNA splicing and inhibition of HIV-1 replication induced by modified U1 small nuclear RNAs.</a> J Virol. 84, 12790-12800 (2010).</li><li>Chang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+expression+and+HIV-1+inhibition+of+chimeric+tRNA(Lys3)-ribozymes+under+dual+U6+snRNA+and+tRNA+promoters.">Enhanced expression and HIV-1 inhibition of chimeric tRNA(Lys3)-ribozymes under dual U6 snRNA and tRNA promoters.</a> Mol Ther. 6, 481-489 (2002).</li><li>Chang, L. J., Liu, X., He, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+siRNAs+targeting+multiple+highly+conserved+RNA+sequences+of+human+immunodeficiency+virus+type+1.">Lentiviral siRNAs targeting multiple highly conserved RNA sequences of human immunodeficiency virus type 1.</a> Gene Ther. 12, 1133-1144 (2005).</li><li>Daniels, S. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=U1+adaptors+result+in+reduction+of+multiple+pre-mRNA+species+principally+by+sequestering+U1snRNP.">U1 adaptors result in reduction of multiple pre-mRNA species principally by sequestering U1snRNP.</a> Nucleic Acids Res. 39, e71 (2011).</li><li>Tai, C. J., Li, C. L., Tai, C. J., Wang, C. K., Lin, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Viral+Entry+Assays+for+the+Identification+and+Evaluation+of+Antiviral+Compounds.">Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds.</a> J Vis Exp. , (2015).</li><li>Riss, T. L., et al., Sittampalam, G. 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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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>[32P]dTTP</td>
			<td>Perkin Elmer</td>
			<td>BLU505H</td>
			<td></td>
		</tr>
		<tr>
			<td>poly(A) RNA template&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>10108626001</td>
			<td></td>
		</tr>
		<tr>
			<td>oligo(dT)12-18 DNA primer</td>
			<td>Thermo Fisher</td>
			<td>18418-012</td>
			<td></td>
		</tr>
		<tr>
			<td>DEAE filtermat paper&#160;</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&#160;</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&#160;</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 ...

Research

JoVE Journal

Immunology and Infection

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

In vitro Uncoating of HIV-1 Cores

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...