key: cord-256561-fnh2do4z
authors: Barik, Sailen; Lu, Patrick
title: Therapy of Respiratory Viral Infections with Intranasal siRNAs
date: 2014-09-23
journal: RNA Interference
DOI: 10.1007/978-1-4939-1538-5_14
sha: 
doc_id: 256561
cord_uid: fnh2do4z

Chemically synthesized short interfering RNA (siRNA) has ushered a new era in the application of RNA interference (RNAi) against viral genes. We have paid particular attention to respiratory viruses that wreak heavy morbidity and mortality worldwide. The clinically significant ones include respiratory syncytial virus (RSV), parainfluenza virus (PIV) (two Paramyxoviruses), and influenza virus (an Orthomyxovirus). As the infection by these viruses is clinically restricted to the respiratory tissues, mainly the lungs, the logical route for the application of the siRNA was also the same, i.e., via the nasal route. Following the initial success of single intranasal siRNA against RSV, we now offer two new strategies: (1) second-generation siRNAs, used against the paramyxoviral RNA polymerase large subunit (L), (2) siRNA cocktail with a novel transfection reagent, used against influenza virus. Based on these results, we propose the following consensus for designing intranasal antiviral siRNAs: (a) modified 19–27 nt-long double-stranded siRNAs are functional in the lung, (b) excessive 2′-OMe and 2′-F modifications in either or both strands of these siRNAs reduce efficacy, (c) limited modifications in the sense strand are beneficial, although their precise efficacy may be position-dependent, (d) cocktail of multiple siRNAs can be highly effective against multiple viral strains and subtypes.

Short interfering RNAs (siRNAs) trigger posttranscriptional gene silencing in a variety of metazoan cells and tissues [ 1 ] . Successful use of synthetic double-stranded siRNA in 2001 [ 1 , 2 ] targeting cellular and viral genes in cell culture, opened the door to siRNA as prospective antivirals and drugs for gene therapy. The "fi rstgeneration" siRNAs were designed to mimic the products of the Dicer endonuclease cleavage; they were 19 nt-long duplexes with 3′-terminal 2 nt overhangs and contained natural, unmodifi ed ribose and bases. As reported by us [ 3 , 4 ] and several other laboratories [ 5 -7 ] , these siRNAs proved effective both in cell culture and in animals. effective in treating these infections, are both relatively nonspecifi c and toxic [ 14 ] . Regarding the molecular features, RSV and PIV are nonsegmented negative-strand RNA viruses, belonging to different genera of the Paramyxoviridae family [ 6 ] . In contrast, fl u is an Orthomyxovirus and contains segmented negative-strand RNA genome [ 9 , 10 ] . Coronoviruses such as SARS-CoV contain positive-strand RNA genomes [ 11 ] . A commonality among these RNA viruses is that they encode genes for RNA-dependent RNA polymerase (RdRP) to transcribe and replicate their own genomes, because the host animal cells are devoid of such activity. Many of the potent antiviral siRNAs have, therefore, targeted viral genes coding for RdRP subunits or proteins that are related to RdRP function [ 3 , 4 , 6 , 9 -11 ] . These early studies targeted: P (Phosphoprotein), L (Large) ( see Table 1 ), and N (Nucleocapsid) genes of RSV; P and L genes of PIV; PA and NP genes of Flu; and the replicase gene of SARS-CoV. In all cases, siRNAs were designed against essential viral gene(s), optimized in cell culture and then used intranasally in the appropriate animal model. Antiviral activity was shown against RSV and parainfl uenza virus (PIV) using the BALB/c mouse model as well [ 4 ] . In those studies, optimal results were obtained with siRNAs delivered in complex with either Subheading 2 ) , although uncomplexed intranasal siRNA also showed some effi cacy [ 4 ] . Intranasal siRNA, complexed with oligofectamine or polyethyleneimine (PEI), was also protective against highly pathogenic infl uenza A viruses of the H5 and H7 subtypes in mice [ 9 , 10 ] . Notwithstanding their success, the activity of these siRNAs was transient, lasting only a few days. Therefore, enhancement of the intracellular and extracellular stability of synthetic siRNAs while increasing (or without compromising) their RNAi activity is a continuing goal for therapeutic translation of RNAi. A variety of chemical modifi cations, including terminal and internal ones, have been added to the fi rst-generation siRNA sequences to improve stability and delivery, leading to what we call "second-generation" siRNAs. Advantage has been taken of the free 2′-OH group of the ribose moiety of RNA (in contrast to DNA that lacks this OH group), to which various substituents were added. We have pursued two promising ones, namely 2′-O-methyl (2′-O-Me) and 2′-fl uoride (2′-F). The latter modification is placed on pyrimidine nucleosides (C, U), leading to 2′-FC and 2′-FU residues. In a number of previous studies [ 16 -19 ] , these substitutions were introduced to various extents in the antisense strand ("guide" strand) or both strands of the siRNA and were shown to enhance stability and potency, although intranasal application was not tested. Additionally, they tend to reduce siRNA-driven innate immune response [ 20 , 21 ] . In systematic studies in cell culture, the biochemical and functional activity of the siRNA was vindicated but found to be affected by the position of the modifi cations in the sequence [ 16 -19 ] . Generally speaking, those with the modifi ed ribonucleotides at the 5′-end of the antisense strand were less active relative to the 3′-modifi ed ones. Internally, while 2′-F was generally well-tolerated on the antisense strand, 2′-O-Me showed signifi cant shift in activity depending on the position. In contrast, incorporation of 2′-O-Me in the sense strand of siRNA did not show a strong positional preference. In a comprehensive study, however, internal 2′-O-Me modifi cations in either or both strands actually made the siRNA less active [ 22 ] . In an animal experiment [ 23 ] , all the 2′-OH residues in siRNAs against hepatitis B virus were substituted with 2′-F and 2′-O-Me. When administered intravenously (i.v.) as lipid complexes, the 2′-O-Me, 2′-F siRNAs showed improved effi cacy and longer halflife in plasma and liver. When these siRNAs were additionally Cy3labeled, it revealed their accumulation in the liver and spleen, but not in the lung, explaining the success of the i.v. administration against hepatitis while suggesting that it is an ineffective route against lung infections.

Based on the absence of a uniform pattern in these studies, we reasoned that siRNAs against respiratory viruses should be individually optimized through the following steps: (1) Design the fi rst-generation siRNA following the generally accepted sequence rules or an algorithm of your choice. (2) Select the ones with lowest IC 50 (preferably below ~20 nM) in a cell culture assay for virus growth, (3) If desired, as we did for RSV (Table 1 ) , add OMe and F substitutions in various "format" (i.e., number and placement) to generate modifi ed, "second-generation" siRNAs. (4) Ensure that the substitutions either improved or did not reduce knockdown effi ciency by screening in cell culture, (5) Confi rm effi cacy and lack of toxicity in animal model, and (6) test improved stability in serum and blood in vitro. Finally, if deemed necessary and resources permit, a low-or high-throughput assay for off-target effects can be performed to further ensure target specifi city.

Together, the small but representative datasets cover multiple viruses, modifi ed and unmodifi ed siRNA. We present a consensus procedure tested in our laboratory, but essentially the same protocol can be used to test other modifi cations or delivery reagents as they become available. In fl u, for example, we have found that the HK polymer is in fact better than a number of other reagents tested (Fig. 1 ).

The reagents described below have been used successfully by us but various equivalents are available commercially that can be optimized. We assume that the reader has access to an appropriate cell culture facility, consisting of incubators and culture hoods, and there is available expertise on virus growth and assay. In this chapter, we will only cover specifi c issues related to the testing of antiviral siRNA against RSV. 3. RNase-free gel-loading microcapillary tips (VWR).

4. RNase-free microfuge tubes (Ambion).

1. Use a free online siRNA design program for your target mRNA sequence ( see Note 1 ). We generally use the one available on the Whitehead Institute (MIT) server at the following URL ( http://sirna.wi.mit.edu/ ); registration is required but free. Copy and paste the target sequence in the box and initially choose AAN 19 TT in the "recommended patterns." Usually, there is no need to change the "Filter criteria" below, i.e., leave them as in the default. Click on the Search button. With RSV L gene, we found many prospective ones (Table 1 ), but if no siRNA is obtained with your gene sequence, repeat the procedure, this time choosing NAN 21 .

With fl u, a proprietary algorithm was used and thus, the siRNA sequences (Fig. 2 ) are also intellectual property. In general, for a wider coverage of multiple virus strains and to minimize resistance, choose viral sequences that are most conserved.

2. This will lead to a table of prospective siRNA sequences that are ordered according to "Thermodynamic values" by the default setting. The goal is to select sequences with high negative values, i.e., sequences closer to the top of the table. In addition, visually examine the central N 19 part (i.e., ignore the fi rst two and the last two nucleotides) and pick the ones that are AT-rich at the 3′-end (right hand side) and GC-rich at the

High effi ciency of the HK polymer in siRNA transfection. A propreitary siRNA against human cyclophilin B mRNA was tested for delivery into HEp-2 cells using the following reagents: His-Lys polymer (HKP) with selected branching [ 27 , 28 ] ; the cationic lipid, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) [ 29 ] ; and 5 % glucose (dextrose) in water (D5W), a common physiological solution used in the clinic. Blank is siRNA administered with no transfection reagent. Note the strong performance of HKP, especially discernible with 10 μg siRNA 5′-end (left hand side). The number of sequences one can test really depends on available resources, but order at least 3 for a given target. Order the sense strand as N 19 dTdT and the antisense strand as (N′) 19 dTdT, such that the N′ part is complementary to the N 19 sequence. Here, we provide an example of tested functional siRNA sequences based on the L polymerase gene of RSV ( Table 1 ). Assume that you will receive the siRNA roughly a week after ordering. Purchase the smallest amount initially (to save money), generally 2 nmol of each. 9. Aliquot the siRNA into small volumes and store at −20 to −80 °C. For best results, do not freeze-thaw more than four times.

1. Trypsinize cells and seed ~5 × 10 4 cells in 500 μl complete growth medium per well of a 24-well plate. Incubate for 24 h to achieve a confl uency of 60-70 %.

2. Immediately before transfection, add 50 μl of Opti-MEM I to a sterile Eppendorf tube. To this, add 2.5 μl TransIT-TKO reagent, mix thoroughly by vortexing, and incubate at room temperature for 10-20 min. Add these volumes to the diluted transfection reagent made in step 2 above and mix by gentle pipetting. Incubate at room temperature for 10-20 min.

4. Adjust the medium volume in each well of the 24-well cell monolayer ( step 1 above) to 250 μl by removing half of the original volume (250 μl). (Table 1 ) . Order the modifi ed siRNA from commercial sources (e.g., Dharmacon) and then process them as in steps 3 -9 (Subheading 3.2 ).

Prior to the intranasal administration of siRNA, the mouse ( see Note 2 ) must be anesthetized by using any standard procedure available in the laboratory, e.g., by administering Nembutal via 

1. Recently, relatively large (26-28 nt) long double-stranded RNAs that act as Dicer substrates (D-siRNA) have been shown to be more potent than the regular 19 nt siRNAs used here [ 24 , 25 ] . We have also found them to be at least as potent as the fi rst and second generation 19-mer siRNA in terms of intranasal anti-RSV activity without increased immune reactions. In preliminary experiments, they also lent well to 2′-O-Me and 2′-F modifi cations for intranasal antiviral activity (data not shown), but it is recommended that the exact format be optimized for each sequence. Follow the published D-siRNA design guidelines [ 24 , 26 ] .

2. Although we have described the laboratory mouse model here, intranasal dosage and delivery can be easily scaled up or down for other laboratory animals.

3. siRNA concentration: The pharmaceutical industry prefers expressing drug concentrations in wt/vol or wt/body weight (e.g., mg/kg). For the researcher, however, it is easier and more useful to express siRNA concentrations in molar units (e.g., μM or nM), since this allows direct comparison between the potency of different siRNAs even when they differ in base composition or modifi cations (and hence formula weight).

4. Transfection reagent: siRNAs may respond differently to different reagents. Experiment with delivery reagent of your choice (Fig. 2 ) .

5. Avoid using excessive liquid because it may suffocate the animal and cause death. As a rule, keep the total instilled volume under 45 μl for BALB/c mice, but higher volumes may be tolerated by larger species.

6. The protocol described here may be modifi ed for aerosolized siRNA using an enclosure to house the anesthetized animal and a handheld nebulizer (the common type used as an inhaler by asthmatics). A larger amount of siRNA is needed because only a fraction of the aerosol is actually inhaled by the animal. If used routinely, consider optimizing a commercial motorized nebulizer. Check with the local pediatricians for the exact model, vendor, and usage. Modify the system by removing the facial mask at the delivery end and inserting the tube directly into the enclosure. A snug fi t of the mask should reduce siRNA waste.

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