key: cord-281254-x7ivjvti
authors: Chang, Zhijie; Babiuk, Lorne A.; Hu, Jim
title: Therapeutic and Prophylactic Potential of Small Interfering RNAs against Severe Acute Respiratory Syndrome: Progress to Date
date: 2012-08-16
journal: BioDrugs
DOI: 10.2165/00063030-200721010-00002
sha: 
doc_id: 281254
cord_uid: x7ivjvti

Severe acute respiratory syndrome (SARS), caused by the novel coronavirus SARS-CoV, produced a scare when it appeared in 2003 in China and later quickly spread to other countries around the world. Although it has since disappeared, its threat to human health remains. Therefore, studies on the prevention and treatment of SARS are important for dealing with epidemics of this and other infectious diseases. The most promising newly developed technology for intervention in SARS may be RNA interference, an endogenous cellular process for the inhibition of gene expression mediated by sequence-specific double-stranded RNAs. Numerous studies have reported the therapeutic potential of RNA interference for the treatment of various human diseases ranging from cancers to infectious diseases such as HIV and hepatitis. To date, most studies on inhibition of SARS-CoV replication using small interfering RNAs (siRNAs) have been conducted in cell lines in vitro. One study using siRNAs to inhibit SARS-CoV infection in Rhesus macaques demonstrated that siRNAs were effective both prophylactically and therapeutically with no adverse effects in the animals. Challenges remaining for the application of siRNA in vivo for SARS prevention and treatment include the specificity of the siRNAs and the efficiency of delivery. However, with improvements in siRNA design and delivery methods, RNA interference has the potential to become another major weapon for combating dangerous infections due to viruses such as SARS-CoV.

During the 2003-4 severe acute respiratory syndrome (SARS) newly developed technology for intervention in SARS may be RNA interference (RNAi).

[10] epidemic, [1] there was a high mortality rate [2] and there were no SARS-CoV, as a member of the coronavirus family, is a singlerational methods for treatment. Empirical practices for treating the stranded plus-sense RNA virus with a membrane envelope. [11] The typical influenza-like symptoms were applied, such as using an-SARS-CoV genome is approximately 30kb in length and encodes tibiotics, antiviral agents (ribavirin, oseltamivir, and HIV-protease a consensus leader sequence, the polyproteins 1a and 1b, and at inhibitors), corticosteroids, interferons, normal human immuleast four major structural proteins including the N (nucleocapsid), noglobulin preparations, and Chinese herbs. [3] Molecular diagnos-S (spike), M (membrane), and E (small envelope) proteins. The tic studies confirmed that a novel coronavirus, named SARS-CoV four structural proteins constitute the virus particle. The N protein (the SARS-associated coronavirus), was responsible for the disis a virion RNA-associated protein and forms a long and flexible ease. [4, 5] This led to the search for rational regimens to treat the helical nucleocapsid; this protein is also important for viral RNA infection, including the generation of vaccines [6] [7] [8] and the design synthesis. The S protein is a glycoprotein on the viral surface and of inhibitors of SARS-CoV replication. [9] The most promising is responsible for viral attachment and entry into host cells. The M protein is necessary for virus budding and the E protein for virus cleavage. In addition, a 3′-hydroxyl group and a 5′-phosphate assembly.

group are generated to enhance the function of the siRNA duplex. [16] While it is simple and quick in its application, this SARS-CoV replication requires first a partial duplication of its chemical synthesis strategy has shown limitations in both stability genome and the translation of some genome-encoded proteins. and production cost of the siRNA. This leads to the first synthesis of a consensus leader sequence, a sub-genome, and the polyproteins 1a and 1b. The leader sequence

To overcome the limitations of chemical synthesis of siRNA, a is capped into the transcripts from the SARS-CoV sub-genome plasmid-based strategy was developed. In this strategy, a dsRNA and plays a role in translation of the structural proteins. [4] of 21 bp with a hairpin loop of nine nucleotides is designed to be Polyproteins 1a and 1b are directly synthesized from the viral expressed from a plasmid under the control of a promoter. [17] In genome without the leader sequence. These proteins include papa-cells, such RNA molecules are subjected to cleavage by Dicer to in-like cysteine protease (PLpro), 3C-like cysteine protease (3CL-generate siRNAs. Since DNA plasmids can be easily and econompro), and RNA-dependent RNA polymerase (RDRP), which are ically produced in Escherichia coli, this strategy has been widely required for viral replication and transcription. [4] Each step in the adopted for the characterization of gene functions in vitro and for key processes during viral replication can be a potential target for therapeutic studies. [14] the interruption of SARS-CoV replication. Since SARS-CoV rep-To address the issue related to delivery of siRNAs into cells or lication also requires certain host proteins, genes from host cells living organisms, researchers have used several approaches, ininvolved in viral replication can also be selected as targets.

cluding: (i) direct introduction of the chemically synthesized siRNAs; (ii) employment of liposome-conjugated siRNAs or plasmids; and (iii) application of viral vectors to express dsRNAs with 1. Small Interfering RNA (siRNA) and its Application a hairpin loop. Each of the methods has both advantages and disadvantages. Recently, numerous papers have reported siRNA RNA interference is an endogenous cellular process for the technology as a potent tool to knock-down gene expression or its inhibition of gene expression mediated by sequence-specific therapeutic potential for treatment of various human diseases double-stranded RNAs (dsRNAs). This phenomenon was first ranging from cancers [18] to infectious diseases (e.g. HIV, hepatiobserved in petunia flowers, and later reported in Caenorhabditis tis). [14] In this review, we focus on the application of siRNA elegans. [12] These dsRNAs, 21-23 bp in length, are produced by technology to SARS. Readers are also referred to another recent cleavage of a long RNA duplex by Dicer, a ribonuclease III-type review on this subject. [19] enzyme. When produced, the small interfering RNA (siRNA) duplexes can activate a process of degradation of mRNA transcripts containing target sequences. This process is driven by the 2. Progress in the Utilization of siRNAs Against the siRNA-enzyme complex or RNA-induced silencing complex Severe Acute Respiratory (RISC) and results in inhibition of gene expression. Interestingly, Syndrome-Associated Coronavirus when dsRNAs are artificially generated either by direct chemical synthesis or by folding single RNA into short dsRNAs with a As the genomic sequence of SARS-CoV was determined and hairpin loop, they can target the specific mRNA sequences and its mechanism of replication became apparent (similar to others in induce degradation of the mRNA. Therefore, RNA interference this coronavirus family), siRNAs targeting different genes of can be a tool for down-regulation of gene expression in cultured SARS-CoV were used by various groups to inhibit virus gene cells as well as in living organisms. [13, 14] expression and thereafter to inhibit replication of the virus (see The two most important issues that must be considered before table I). The initial report came from two groups using the chemithe application of siRNAs to down-regulation of gene expression cal synthesis strategy. [20, 21] The first group showed that two of six becomes a reality are: (i) generation of effective dsRNAs; and (ii) chemically synthesized siRNAs, based on the SARS-CoV genome delivery of the dsRNAs into targeted cells or organs. To address sequence, resulted in the inhibition of viral cytopathic effect (CPE) the first issue, researchers have established two strategies. The in Vero-E6 cells infected with strain Y3-SARS-CoV. [21] The curfirst one is to chemically synthesize siRNA. [15] In this method, two sory evaluation of the CPE of the transfected cells provided single-stranded, complementary, short RNA oligonucleotides are evidence that siRNA might be effective in inhibiting SARS-CoV synthesized. These two short oligonucleotides are then annealed replication. The second group demonstrated that three of six into a dsRNA in a test tube. Usually short oligonucleotides (21) (22) (23) siRNAs designed to target the replicase A of SARS-CoV were nucleotides) are designed with a two-nucleotide overhang to facili-moderately effective, whereas the other three were less effectate the activation of the RISC during the process of mRNA tive. [20] Around at the same time, Zhang et al. [22] used a vector-based potential tools to inhibit SARS-CoV replication. Even though the strategy and designed two different siRNAs targeting the se-S protein has a high mutation rate, [36] targeting its mRNA has also quences encoding the S protein of SARS-CoV. These sequences been reported by other groups to be effective in the inhibition of were chosen because they were identical in different SARS-CoV SARS-CoV replication. [25, 27] strains. The authors inserted the siRNA sequences into the pBS/U6

Meanwhile, Wang et al. [23] adopted a similar strategy by targetvector with a U6 promoter, which expresses siRNAs in cells. This ing the viral RNA polymerase gene of SARS-CoV. This group group of researchers was the first to demonstrate that the siRNAs used a commercially available vector pSUPER.retro and designed inhibited S gene expression and virus replication in Vero E6 cells six siRNAs. These researchers observed that in Vero E6 cells infected with SARS-CoV strain BJ01. This study provided strong infected with the SARS-CoV strain HKU-39849, two of the six evidence that siRNAs targeting the S gene could be employed as siRNAs had a dramatic effect on the inhibition of viral replication.

replication than siRNAs targeting the S gene. Later, Ni et al. [28] The effectiveness of targeting the RNA polymerase gene was selected three of 32 potential siRNA sequences targeting the further confirmed by Lu et al. [26] and Meng et al. [35] leader sequence. They demonstrated that three siRNAs delivered in the pSilencer3.0-H1 vector dramatically inhibited SARS-CoV To avoid the tedious work of constructing plasmid vectors to replication as measured by either plaque formation or levels of test effective target sequences, Zheng et al. [31] chemically synthe-mRNAs and proteins (S and N). While another group did not find sized 48 siRNA duplexes targeting regions throughout the entire any significant inhibitory effect of the chemically synthesized SARS-CoV RNA genome, including the open reading frames for siRNA duplex targeting the leader sequence, [27] Li et al. [24] and Ni several key proteins. They transfected these siRNA duplexes into et al. [28] have separately demonstrated the effectiveness of vectorfetal Rhesus kidney (FRhK-4) cells prior to, or after, infection with driven siRNAs targeting the leader sequence. Whether there is a the virus, and evaluated the efficiency of the siRNAs on inhibition difference between the vector-driven and the chemically syntheof viral replication. They found that four of the siRNA duplexes sized siRNAs remains unclear. had a potent inhibitory effect on SARS-CoV infection and replication, evaluated by intracellular viral genome copy number and

The rational design for effective siRNA inhibition of SARSviral titers in the cell culture medium using quantitative reverse

CoV replication remains difficult, as most authors have selected transcription PCR (Q-RT-PCR) and CPE-based titration, respectarget sequences mainly by empirical testing. The selected target tively. In this study, a prolonged prophylactic effect of siRNA sequences usually have some specific features, as previous reports duplexes with up to 90% inhibition that lasted for at least 72 hours have suggested. [14, 39] For the ease of selection of siRNA target was observed. Combination of active siRNA duplexes, targeting sequences, Meng et al. [35] adopted a cell-based assay to screen for different regions of the viral genome, resulted in up to 80% siRNAs to block SARS-CoV gene expression. This assay involved inhibition in vitro.

two genes encoding RDRP and the envelope E protein, respectively. These authors verified the published sequences and validated Because they are convenient and rapid to generate, the chemitwo siRNA sites for the RDRP gene and two sites for the envelope cally synthesized siRNA duplexes seem attractive for the identifi-E gene. This assay provided a specific strategy to select effective cation of targets. Following the study by Zheng et al., [31] Shi et siRNA sequences. al. [30] synthesized 26 siRNA duplexes and showed that three of these effectively inhibited the expression of a reporter gene carry-Considering that the virus may possibly escape from siRNA ing a target sequence in Vero E6 cells. To improve the effectivetargeting through viral mutations, [40, 41] He et al. [33] used a combiness of inhibition, this group adopted a strategy utilizing a mutated nation of siRNAs targeting different sequences. These authors nucleotide at the last pair of the duplex (with a TT overhang). They reduced the dosage of the single siRNA duplexes to a minimal found this modification of the duplex increased the inhibitory effective level and then tested the synergistic effect of the combieffects on SARS-CoV gene expression. This observation connation of the different duplexes. They found that the combination firmed that a mismatch in the 5′ end of the antisense siRNA can of siRNAs targeting two different sites had a significant synergisenhance its effectiveness. [37, 38] tic effect on the inhibition of SARS-CoV replication in infected FRhk-4 cells. However, it is still unclear as to whether this Recently, more siRNAs to inhibit SARS-CoV gene expression synergistic role could overcome the problem of viral escape and replication have been reported. Wu et al. [27] described three through mutation. In this study, they did not observe any synergischemically synthesized siRNA duplexes targeting the S gene and tic effect of the combination of the siRNA duplexes at high dose, the 3′-UTR sequences (si-SARS-S2, siSARS-S3, and siSARalthough this may be explained by a saturated dosage effect. [20, 31] S-3′UTR2). Zhao et al. [29] designed several siRNAs targeting the sequence encoding the N protein and, more recently, Meng et Currently, researchers have used both chemical synthesis and al. [35] identified siRNAs targeting the E and RDRP genes.

vector-based approaches to generate siRNAs for SARS-CoV. Interestingly, two groups have reported the effects of targeting While the chemical synthesis approach is more rapid and easy to the leader sequence of SARS-CoV. [24, 28] Li et al. [24] first identified test, the RNA stability is problematic. To improve the biostability, the same leader sequence in the mRNA encoding different struc-Elmen et al. [34] used a locked nucleic acid (LNA) approach for the tural proteins. Based on this finding, they reasoned that siRNAs design of siRNAS. As reported by Braasch et al., [15] LNA could targeting the leader sequence should effectively inhibit viral repli-increase the thermal stability of siRNA duplexes without any cation in cell culture systems, since they would target a number of deleterious effect on their function. The study by Elmen et al. [34] proteins simultaneously. This research group provided evidence indicated that incorporation of LNA significantly enhanced siRNA that siRNA targeting the leader sequence, via a U6 promoter-based stability and improved the efficiency of the unmodified siRNA on vector, had a stronger effect on the inhibition of SARS-CoV SARS-CoV inhibition. This study demonstrated that the modifica-tion of the siRNA duplex could substantially improve its activity. siRNAs targeting different sequences. This had a further beneficial Therefore, this approach may enhance the development of siRNAs effect since it appears that the combination of the different siRNAs as a therapeutic intervention for SARS-CoV.

had a synergistic effect on the inhibition of SARS-CoV replication. Based on this data, it appears that the best approach would be To date, most studies on the inhibition of SARS-CoV replicato use a combination of siRNAs for maximal benefit; therefore, we tion using siRNAs have been conducted using cell lines in vitro.

suggest that highly effective siRNAs in combination should be However, in the only animal study to date, Li et al. [32] have used. reported their attempts at using siRNAs to inhibit SARS-CoV infection in Rhesus macaques. After initial screening, two chemi-One possible adverse effect of in vivo application is that some cally synthesized siRNAs were selected from 48 siRNAs based on siRNAs may trigger host immune responses such as the induction the previous work of Zheng et al. [31] The researchers treated the of interferon. [42] However, since most siRNAs do not show this animals with the two siRNA duplexes in a commonly used glucose adverse effect, it is possible to conduct screening of siRNA solution D5W prior to, and after, infection with SARS-CoV and sequences in animals before undertaking clinical studies. Another found that all of the monkeys in the treated groups showed less potential problem with the application of this technology in vivo is severe symptoms than those in the control groups. They reported that when siRNAs are overexpressed, they may interfere with that during a 20-day period of observation after the administration endogenous microRNA processing. [43] This problem can be avoidof the siRNA duplexes, the treated monkeys showed an average ed by lowering the delivery dose or siRNA expression. body temperature of <38.9°C, while the control animals had an average body temperature of >39.1°C. The temperature changes 3.2 Delivery of siRNA were correlated with the changes in lung histopathology. The intriguing observation was that three of the four animals in each of All the studies referred to in this review have demonstrated the three siRNA-treated groups were free of SARS-CoV in the potent inhibitory effects of siRNAs (both chemically synthesized oropharyngeal swab specimens, based on measurements of SARSand vector based) on the replication of SARS-CoV in cultured CoV RNA copy numbers. This indicated that the SARS-CoV cells or in animals. However, for clinical applications in both the particles released from the animals' lungs were dramatically reprophylactic and therapeutic contexts, it is critical to improve the duced by the injection of the siRNA duplexes. Whether siRNA efficiency of siRNA delivery into specific human organs. As in the inhibited SARS-CoV replication or blocked the spread of the virus gene therapy field, [44] siRNA targeting SARS-CoV is hampered by within the lungs remains to be determined. However, this study barriers to delivery. In the work done in Rhesus macaques, Li et clearly demonstrated that siRNAs were effective both prophylactial. [32] adopted a very common glucose solution D5W to deliver the cally and therapeutically with no adverse effects in the animal chemically synthesized siRNAs into the airways of animals. Alstudy. [10] The reduction of virus in the lungs may dramatically though the experimental results demonstrated dramatic changes in reduce spread to contacts, thereby being an effective approach to SARS-CoV replication, it remained unclear how much of the reducing spread of the virus between patients and caregivers. siRNA duplexes had been successfully delivered into the airway epithelial cells of the animals, and how long the in vivo transfected 3. Challenges to In Vivo Application of siRNA siRNA duplexes were maintained in the cells. Clearly, this is an area where additional work is needed. However, we are confident Two challenges remain for the application of siRNA in vivo for that with all the recent work in gene targeting and vaccine formu-SARS prevention and treatment. These involve the specificity of lation, this hurdle is not insurmountable. the siRNAs and the efficiency of their delivery.

Approaches for gene delivery to airways have been greatly improved over the past decade, especially those using viral vec-

tors, such as the helper-dependent adenoviral vector (HDAd) The target specificity of siRNA is an important feature of this which can transduce almost all the airway epithelial cells in mice novel technology for therapeutic development. Highly specific and to a lesser degree in rabbits. [45] [46] [47] Delivery of siRNAs with siRNAs can be selected through screening to avoid potential off-viral vectors should be more effective for prophylactic use, since target side effects. However, this high specificity can also be a expression of siRNAs from viral vectors is expected to last much problem since a single nucleotide change will diminish the effec-longer than the duration of chemically synthesized siRNA in tiveness of the designed siRNA. Since mutations of the SARS-transfected cells. Although viral vectors can be used to deliver CoV genome can occur, [36] which may result in viral escape from siRNAs for SARS-CoV, potential adverse effects, such as inflam-siRNA targeting, [40, 41] He et al. [ example, Cao et al. [48] have shown that interleukin (IL)-8 expres- 

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Locked nucleic acid (LNA) mediated dependent adenovirus formulation to rabbit lung using an intratracheal catheter. improvements in siRNA stability and functionality

Readministration of helper-dependent

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Down-regulation of IL-8 expression in human

Comparative full-length genome sequence analysis airway epithelial cells through helper-dependent adenoviral-mediated RNA of 14 SARS coronavirus isolates and common mutations associated with interference

Schiffelers RM, Storm G. ICS-283: a system for targeted intravenous delivery of

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Correspondence: Dr Jim Hu, Lung Biology Research Program

ON M5G 1X8, Canada. and passage of SARS coronavirus in cells from non-human primates