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RNA interference- New Drug Discovery for Hepatitis B Therapy
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"RNA interference-mediated control of hepatitis B virus and emergence of resistant mutant"
"...It is striking how rapidly the field has moved from an initial discovery phase to a stage where implementation and evaluation of siRNA technology in a therapeutic setting is expected sometime this year......The next few years of basic and clinical research will indicate whether RNAi technology will realize its full potential as the "next wave of truly novel therapeutic molecules...."
FOLLOWING THIS STUDY REPORT IS
Editorial called: "Therapies for Hepatitis B: where do we go from here"
Gastroenterology March 2005, Vol 128, Num 3
Hui-Lin Wu
Hepatitis Research Center, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
With an estimated 300 million chronic carriers, hepatitis B virus (HBV) infection remains one of the most prevalent chronic viral infections of humans.1 Chronic infections cause serious consequences, including cirrhosis and hepatocellular carcinoma,2 responsible for at least 1 million deaths annually worldwide.1 Thus, control of HBV infection by prevention or treatment is imperative.
A preventive vaccine is available to block HBV infection successfully; however, therapeutic intervention is the only option for chronic HBV carriers to delay or prevent the progression to life-threatening, end-stage liver diseases. Immune modulators (ie, interferon [IFN-]) and nucleoside analogues (ie, lamivudine) are 2 approved mainstream treatments for chronic hepatitis B. However, both therapies achieve only limited response, and none of them can eradicate the virus effectively.3 Other strategies, including antisense RNA and DNA constructs, hammerhead ribozyme, and dominant negative HBV core proteins, are still under development.4,5 Therefore, the quest for potent antiviral medications to treat chronic hepatitis B continues.
Recently, it was found that double-stranded RNA (dsRNA) can trigger a sequence-specific, gene-silencing process called RNA interference (RNAi).6 RNAi is a very specific and potent mechanism to silence gene expression through processing of double-stranded RNA into 2126 nucleotide (nt), short interfering RNA (siRNA). In plants, it serves as a host-defense mechanism against viruses and transposable elements (reviewed in Waterhouse et al7). After the discovery that RNAi pathways also exist in mammalian cells and that the use of siRNA can avoid cell death induced by dsRNA longer than 30 nucleotides,8 siRNA has emerged as a novel therapeutic approach in the fight against human viral infections. This study therefore examined whether siRNA can inhibit HBV replication and expression and exhibit the potential to combat chronic hepatitis B.
During the course of this work, a growing number of studies have shown that siRNA can be effective against several pathogenic viruses, including poliovirus, HIV, HCV replicons, influenza virus, SARS coronavirus, and others.9,10 Several studies employing siRNA or short hairpin RNA (shRNA) to inhibit HBV1118 also revealed that RNA interference indeed has the potential to treat viral infections, including HBV. However, variation of sequences among different viral isolates in clinical settings and emergence of resistant mutants constitute potential problems hindering the efficacy of siRNA. This work identified a target site for siRNA that is conserved among HBV genotypes A to G. A plasmid expressing shRNA targeted this site significantly inhibited the steps in HBV replication that occur in cultured cells and in mice. The problem of resistant mutants was also explored in this study.
ABSTRACT
Background & Aims: Present therapy for chronic hepatitis B attains control only in limited proportions. Small interfering RNA (siRNA) offers a new tool with potential therapeutic applications for hepatitis B virus (HBV). Given the importance of sequence identity in the effectiveness of siRNA and the heterogeneity of HBV sequences among different isolates, a short hairpin RNA (shRNA)-expressing plasmid, pSuper/HBVS1, was developed to target a region conserved among major HBV genotypes and assess its effectiveness control of HBV.
Methods: HBV replication-competent plasmid was cotransfected with pSuper/HBVS1 to HuH-7 cells or to mice. The levels of viral proteins, RNA, and DNA were examined in transfected cells and animals. The effects of pSuper/HBVS1 on clinical isolates with genotypes B and C were also determined.
Results: pSuper/HBVS1 significantly decreased levels of viral proteins, RNA, and DNA for HBV genotype A in cell culture and in mice. Comparable suppressive effects were observed on clinical isolates of genotypes B and C. A clone with a silent mutation in the target region was identified from a patient with genotype C. This mutant revealed diminished sensitivity to pSuper/HBVS1 and could be selected out in the presence of pSuper/HBVS1 in cell culture.
Conclusions: These findings indicated that shRNA could suppress HBV expression and replication for genotypes A, B, and C, promising an advance in treatment of HBV. However, the emergence of resistant mutants in HBV quasispecies should be considered.
AUTHOR DISCUSSION
With the advantages of being highly specific, potent, and safe for gene silencing, the potential of using siRNA as an antiviral agent has been actively explored in numerous studies. In this work, RNA polymerase III promoter-based vector was used to express shRNA against HBV and clearly demonstrated the strength of this vector in inhibiting HBV expression and replication in both cell culture and in mouse model systems. Furthermore, the shRNA-expressing plasmid used in this study cannot only suppress the expression of laboratory prototype HBV strain but also can suppress expression of HBV from clinical isolates with different genotypes.
During the preparation of this work, several studies documented the anti-HBV activity of RNA silencing by either synthetic siRNA or shRNA-expression plasmids in vitro and in vivo. These results resembled the present investigative outcomes while supporting siRNA as a promising approach of controlling HBV. What makes pSuper/HBVS1 distinctive is that its target site is the only one conserved for 7 HBV major genotypes (A to G) among all siRNA target sequences published so far. Only genotype H contains a nucleotide difference within the pSuper/HBVS1 target sequence. Nucleotide 472 (the same nucleotide in the previously identified pSuper/HBVS1 resistant clone) is G in genotype H rather than T in other genotypes. Nevertheless, genotype H is not common worldwide. Given the high heterogeneity of HBV sequences and the sensitivity of siRNA to the changes of sequences, it is advantageous to design potent siRNA targeting highly conserved regions among various HBV isolates. However, it is very challenging to develop an siRNA targeting highly conserved regions and also being very effective for HBV. In fact, pSuper/HBVS1 is the only one found so far. Conservation of an siRNA target sequence can generalize the utility of siRNA to different HBV genotypes and strains, as illustrated by the suppressive effects of pSuper/HBVS1 on the expression of HBV genotypes A, B, and C in this study. Although the inhibitory effects of pSuper/HBVS1 on other genotypes were not tested because of lack of clinical samples, pSuper/HBVS1 very likely can suppress the expression of other genotypes with the conserved target sequence.
Although this and other studies have proved the feasibility of using siRNA to control HBV expression and replication in vivo and in vitro, emergence of resistant mutants may be problematic. Previous studies on using siRNA to control replication of human immunodeficiency virus or poliovirus in culture have noted the emergence of resistant mutants. These resistant mutants either emerged de novo with molecularly cloned viral genomes29,30 or were likely present in the initial virus population and selected out in the presence of siRNA. In this study, an HBV variant with a silent mutation within the target site of pSuper/HBVS1 was found from one HBV chronic carrier. To our knowledge, this is the first experimental proof that resistant mutant indeed preexists in the quasispecies of HBV patients. Data were also presented supporting the notion that this mutant can be selected out in the presence of siRNA. This finding, together with others, underlines the importance of sequence identity of siRNA to its target sequence to be effective. Combinations of multiple siRNA targeting separate regions of the genome can alleviate the problem of resistant mutants. Use of siRNA targeting functionally indispensable and conserved regions can further minimize this problem, reducing the numbers of siRNA used and therefore the cost. For pSuper/HBVS1, the mutation identified for pHBV-CI-m at nucleotide 472 caused silent mutation for both surface and polymerase genes within this conserved target region. In genotype H, the difference at nucleotide 472 left unchanged the amino acid sequence of HBV surface proteins, causing Leu to Val conservative change in the polymerase reading frame. These findings implied that probably only very few mutations can be tolerated by HBV within pSuper/HBVS1 target sequence. Therefore, preventing the emergence of resistant clones can be simply achieved by including shRNA against the mutant sequence. This problem may also be overcome by combining with other antiviral strategies or siRNA targeting host factors that are dispensable for cell but essential for virus.
Because all HBV transcripts overlap in their C terminals, a single shRNA targeting multiple critical transcripts simultaneously could be designed to maximize the inhibitory potency. In this regard, pSuper/HBVS1 targets all HBV major transcripts except X mRNA. Interestingly, this investigation found that pSuper/HBVS1 suppressed the expression of HBsAg more efficiently than that of HBeAg, although it targeted the same sequence on the transcripts for these 2 proteins. In contrast, the S-target siRNA used by Klein et al resulted in greater suppression of HBeAg than HBsAg. Song et al also noticed that the same siRNA could have varying suppressive effects against human immunodeficiency virus in different cell types. Currently, it is uncertain why siRNA exerts differential suppressive effects on the same target sequence on different RNA molecules or in different cell types. One possible speculation is that the target sequence in different RNA molecules may have different secondary structures or are bound with different proteins and thus make their accessibility to the same siRNA different.
In natural infections, HBV uses covalently closed circular DNA (cccDNA) as the template for transcription. Therefore, to treat chronic HBV infection, the siRNA needs to either sustain in the infected hepatocytes to continuously degrade HBV RNA or to inactivate cccDNA from transcription. Thus far, siRNA-induced gene silencing in mammalian cells seems to be transient because of the likely lack of the RNA silencing-amplification mechanism present in worms and plants. Recently developed lentivirus, retrovirus, and adenovirus-associated virus (AAV)-based shRNA expression vectors may provide a promising approach for long-term expression in mammals, although safety issues would be a concern. The effect of siRNA/shRNA on cccDNA of HBV would not be easy to address in the experimental systems used in the current study. Neither HCC cells transfected with HBV DNA nor mice hydrodynamically injected with HBV DNA have been generally proficient in providing cccDNA templates for pregenomic RNA synthesis. Recent advances in inducing DNA methylation by siRNA in human cells have shed light on using siRNA to inactivate HBV cccDNA from transcription. Further studies will address the effects of siRNA on HBV cccDNA.
This and other studies have clearly established that siRNA/shRNA could serve as a powerful potential therapeutic tool for treating hepatitis B infection as well as other diseases, although some technical obstacles must be overcome. The quick advances of siRNA technology, including optimization of target sequences, improvement of delivery methods, and expression vectors, would definitely render siRNA more clinically feasible and useful in the future. Furthermore, more knowledge of the working mechanisms of the siRNA-mediated effect on DNA genome and systemic silencing in plants and other organisms (reviewed in Agrawal38) would facilitate the development of strategies to induce these activities in mammalian cells. By then, siRNA will have become an even more potent and ideal therapeutic agent for HBV as well as other viral infections.
Editorials
Therapies for hepatitis B: Where to from here?
Stephen Locarnini
Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, Australia
Over the last 5 years, the clinical management of hepatitis B has experienced a renaissance. This has been mainly due to the introduction, evaluation, and approval of a number of nucleoside/nucleotide analogues with specific antiviral activity against the hepatitis B virus (HBV). By the end of 2004 in most countries, 3 therapies have been approved for the treatment of chronic hepatitis B (CH-B): interferon-alpha, lamivudine, and adefovir dipivoxil.
In the last few years, the results of a number of excellent clinical trials have been published using various combinations of these agents. With this amount of clinical trial activity, one would be anticipating substantial progress in patient management. Indeed, progress has been good, a lot has been learned, but significant challenges still remain. The majority of patients being treated with the approved therapies still do not reach a satisfactory clinical end-point, at least in the short term. Therapeutic efficacy, durable viral suppression, drug resistance, patient monitoring, tolerability, and patient compliance are now everyday issues for the treating clinician. In essence, treating patients with CH-B is hard.
The list of possible host and virological factors associated with the relatively low response rate of existing approved therapies includes the recalcitrant nature of the HBV covalently closed circular (ccc) DNA replicative intermediate in the liver, inadequate, and/or inappropriate host immune-responses, the persisting toleragenic effects of the hepatitis Be antigen and the role of excess hepatitis B surface antigen circulating in blood. Most investigators agree in principle that a combination of some form of immune modulation/manipulation and directly acting antiviral agent, preferably virocidal rather than virostatic, is now required to adequately treat and control CH-B. Unfortunately to date, the results of combinations of interferon and nucleoside/nucleotide analogues have been disappointing.1 So where do we go from here? Clearly, new approaches are needed and in this issue of GASTROENTEROLOGY Hui-Lin Wu et al.2 describe the control of HBV replication by the process of RNA interference (RNAi), using short interfering (si)RNA. So what is RNAi and siRNA?
Introducing RNA silencing
RNA silencing or posttranscriptional gene (PTG) silencing, is the process whereby double-stranded RNA (dsRNA) induces the sequence-specific degradation of homologous messenger RNA (mRNA). The current model of RNAi involves a multi-step process.3,4 First, the initial dsRNA (>26bp) is cleaved by an RNAse III-like enzyme, termed Dicer, to generate 2123nt fragments of siRNA. Second, an ATP-dependent RNA helicase recognizes these short duplexes and resolves the siRNA duplex into 2 single-stranded RNAs. One strand is then incorporated into a high-molecular-weight protein complex termed RISC (RNA-induced silencing complex), where it serves as guide RNA to direct the cleavage of homologous RNA sequences by an endonucleolytic component of RISC. The RISC is liberated from the cleaved mRNA and is recycled to perform multiple rounds of catalysis.3,4
RNA silencing was initially discovered in plants more than a decade ago. Although RNAi is evolutionarily conserved among plants and animals,5 studying silencing of specific genes in mammalian cells has been difficult because of the induction of the interferon response by dsRNAs of 30 base pairs (bp).3 This well-known antiviral interferon response globally represses mRNA translation,6 causing substantial changes in cellular gene expression, resulting in the induction of apoptosis, and thereby masking any specific silencing effect by RNAi in mammalian cells. However, it has been shown recently that potent and specific gene silencing can be achieved in human cells transfected with siRNAs of 2123nt and that these are the key intermediates in the RNAi pathway.
RNAi-mediated inhibition of viral replication
In plants, PTG silencing and RNAi play critical roles in genome surveillance, protecting the cell from inappropriate expression of repetitive sequences and transposable elements, as well as a major antiviral defense mechanism.8 The ability to tap into this innate antiviral pathway to devise possible treatments against viruses is rapidly emerging as a new field of therapeutic intervention. To date, RNAi has been used effectively to inhibit the replication of several different pathogenic human viruses in culture, including respiratory syncytial virus (RSV),9 poliovirus,10 papillomavirus,11 HIV-1,12 hepatitis C virus (HCV),13 and HBV.14,15 In addition to targeting viruses directly, complementary strategies have used siRNAs that silence the expression of essential host factors required for productive viral replication including the chemokine receptor CCR5, a co-receptor for HIV-1 cell entry,16 the CD4 molecule, the principal receptor for HIV-112 and Tsg101, which is required for vacuolar sorting and efficient budding of HIV-1 progeny.17
Using RNAi as an anti-HBV tool has several important advantages. Specifically targeting the viral transcripts severely impairs HBV replication, without activating nonspecific cellular responses, hence minimizing undesirable side effects and cell toxicity associated with increased activity of 2, 5 oligoadenylate synthetase and protein kinase R.6 Targeting conserved regions in the HBV genome should reduce the selection of escape mutants and the potential to introduce several siRNAs targeted to different sequences simultaneously will further reduce this risk and make it possible to treat patients infected with diverse circulating HBV genomes.2,14 The ability of siRNAs to reduce the levels of viral transcripts and proteins by a mechanism independent of inhibition of reverse transcription and active HBV genomic replication, makes it a good candidate for combination therapy with nucleoside/nucleotide analogues and/or the interferons. Finally, the nature of the HBV genome with its use of overlapping reading frames means that multiple viral mRNAs can be inhibited by a single siRNA.2
Several studies have now been reported describing the inhibiting effects against HBV by RNAi in cell culture14,15,18 and in vivo15,19 and in general, impressive effects have been observed. However, the region of the HBV genome targeted for silencing is an important determinant of efficacy. Klein et al.15 demonstrated that siRNAs targeted to different sequences in the envelope open-reading frame varied in their overall inhibiting effect on gene expression. Target sequences with the most consistent and effective silencing seem to be directed to regulatory elements such as the unique polyadenylation signal sequence of HBV18 or highly conserved target sequences in the viral envelope gene.2
Clinical implementation of RNA interference
Delivery
A major impediment to using RNAi technology for therapeutic benefit has been the development of efficient delivery systems for siRNAs. However, several companies are developing clinically appropriate approaches to overcome this obstacle (see Nucleonics Inc. at www.nucleonicsinc.com). Previous methods relied on harsh lipid-based transfection reagents to introduce siRNAs into cells in culture and are either inefficient and/or unsuitable for use in animals. Interestingly, injected siRNAs have been shown to have a high affinity for the liver.19,20 A recent study has indicated that approximately 90% of hepatocytes are reached after siRNA transfer via hydrodynamic tail vein injection in mice, demonstrating that the liver is a preferred target organ for siRNA delivery.21
An important step forward in addressing these complications has been the finding that natural or designed siRNAs can be expressed in vivo.2225 Short hairpin-based precursor RNAs can be transcribed from either RNA polymerase III22,24 or RNA polymerase II promoters25 in vivo, and processed by Dicer to release functional siRNAs. These siRNAs expressed from DNA templates can silence gene expression as effectively as exogenously introduced synthetic siRNAs.22,25 Many groups have constructed retroviral-based,26 adenoviral-based,27 and lentiviral-based28 gene therapy vectors that are capable of expressing siRNAs in a stable manner in virtually any cell and tissue type. Substantial progress has been made already, with the demonstration that murine retroviral vectors expressing siRNAs directed against a mutant allele of the human K-ras proto-oncogene have the ability to reverse tumorigenicity in mice.26 The further development of these delivery systems are driving siRNA technology quickly from the "proof of principle" phase into animal studies of important human diseases and into early clinical trials.
Resistance
The selection of resistant strains of poliovirus10 and HIV29 in cultures treated with specific siRNAs is a reminder that a single nucleotide mutation is all that is needed to allow the virus to escape the effect of a single specific siRNA. Emergence of siRNA resistance is a major concern that will need to be addressed, particularly for viruses encoding error-prone polymerases such as those of HIV-1 and HBV. In the study of Wu et al,2 sequence analysis of an HBV clinical isolate showed that a change of T to C at nucleotide 472 resulted in RNAi escape. Thus, siRNA expression cassettes will require the ability to encode tandem arrays of highly expressed siRNAs that target several conserved viral sequences simultaneously.
Conclusions and future challenges
It is striking how rapidly the field has moved from an initial discovery phase to a stage where implementation and evaluation of siRNA technology in a therapeutic setting is expected sometime this year. However, our understanding of the biological mechanisms underlying RNAi lags behind the momentum to apply this technology to human diseases such as cancer and infectious diseases caused by HIV-1, HCV, and HBV. Clearly the objective, in the short term, is to improve viral delivery systems with the goal of maximizing siRNA expression. Presumably, this would involve optimization of promoters and siRNA precursor design. A better understanding of the fundamental biochemistry of the RNAi pathway is still required and would certainly lead to improved target site selection and better overall siRNA design. The next few years of basic and clinical research will indicate whether RNAi technology will realize its full potential as the "next wave of truly novel therapeutic molecules."8
Editorial References
1. Marcellin P, Lau GK, Bonino F, Farci P, Hadziyannis S, Jin R, Lu ZM, Piratvisuth T, Germanidis G, Yurdaydin C, Diago M, Gurel S, Lai MY, Button P, Pluck N. Peginterferon alfa-2a alone, lamivudine alone, and the two in combination in patients with HBeAg-negative chronic hepatitis B N Engl J Med 2004;351:1206-1217.
2. Wu H-L, Huang L-R, Huang C-C, Lai H-L, Liu C-J, Huang Y-T, Hsu Y-W, Lu C-Y, Chen D-S, Chen P-J. RNA interference-mediated control of hepatitis B virus and emergence of resistant mutant Gastroenterology 2005;128:708-716.
3. McManus MT, Sharp PA. Gene silencing in mammals by small interfering RNAs Nat Rev Genet 2002;3:737-747.
4. Hutvagner G, Zamore PD. RNAi: nature abhors a double-strand Curr Opin Genet Dev 2002;12:225-232.
5. Fire A, Xu S-Q, Montgomery M, Kostas S, Driver S, Mello C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans Nature 1998;391:806-811.
6. Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD. How cells respond to interferons? Annu Rev Biochem 1998;67:227-264.
7. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells Nature 2001;411:494-498.
8. Coburn GA, Cullen BR. siRNAs: a new wave of RNA-based therapeutics J Antimicrob Chemother 2003;51:753-756.
9. Bitko V, Barik S. Phenotypic silencing of cytoplasmic genes using sequence-specific double-stranded short interfering RNA and its application in the reverse genetics of wild type negative-strand RNA viruses BMC Microbiol 2001;1:34.
10. Gitlin L, Karelsky S, Andino R. Short interfering RNA confers intracellular antiviral immunity in human cells Nature 2002;418:430-434.
11. Jiang M, Milner J. Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference Oncogene 2002;21:6041-6048.
12. Novina CD, Murray MF, Dykxhoorn DM, Beresford PJ, Riess J, Lee SK, Collman RG, Lieberman J, Shankar P, Sharp PA. siRNA-directed inhibition of HIV-1 infection Nat Med 2002;8:681-686.
13. Kapadia SB, Brideau-Andersen A, Chisari FV. Interference of hepatitis C virus RNA replication by short interfering RNAs Proc Natl Acad Sci U S A 2003;100:2014-2018.
14. Shlomai A, Shaul Y. Inhibition of hepatitis B virus expression and replication by RNA interference Hepatology 2003;37:764-770.
15. Klein C, Bock CT, Wedemeyer H, Wustefeld T, Locarnini S, Dienes HP, Kubicka S, Manns MP, Trautwein C. Inhibition of hepatitis B virus replication in vivo by nucleoside analogues and siRNA Gastroenterology 2003;125:9-18.
16. Martinez MA, Gutierrez A, Armand-Ugon M, Blanco J, Parera M, Gomez J, Clotet B, Este JA. Suppression of chemokine receptor expression by RNA interference allows for inhibition of HIV-1 replication AIDS 2002;16:2385-2390.
17. Garrus JE, von Schwedler UK, Pornillos OW, Morham SG, Zavitz KH, Wang HE, Wettstein DA, Stray KM, Cote M, Rich RL, Myszka DG, Sundquist WI. Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding Cell 2001;107:55-65.
18. Konishi M, Wu CH, Wu GY. Inhibition of HBV replication by siRNA in a stable HBV-producing cell line Hepatology 2003;38:842-850.
19. McCaffrey AP, Nakai H, Pandey K, Huang Z, Salazar FH, Xu H, Wieland SF, Marion PL, Kay MA. Inhibition of hepatitis B virus in mice by RNA interference Nat Biotechnol 2003;21:639-644.
20. Lewis DL, Hagstrom JE, Loomis AG, Wolff JA, Herweijer H. Efficient delivery of siRNA for inhibition of gene expression in postnatal mice Nat Genet 2002;32:107-108.
21. Song E, Lee SK, Wang J, Ince N, Ouyang N, Min J, Chen J, Shankar P, Lieberman J. RNA interference targeting Fas protects mice from fulminant hepatitis Nat Med 2003;9:347-351.
22. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells Science 2002;296:550-553.
23. Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells Genes Dev 2002;16:948-958.
24. Paul CP, Good PD, Winer I, Engelke DR. Effective expression of small interfering RNA in human cells Nat Biotechnol 2002;20:505-508.
25. Zeng Y, Wagner E, Cullen B. Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells Mol Cell 2002;9:1327-1333.
26. Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference Cancer Cell 2002;2:243-247.
27. Xia H, Mao Q, Paulson HL, Davidson BL. siRNA-mediated gene silencing in vitro and in vivo Nat Biotechnol 2002;20:1006-1010.
28. Qin XF, An DS, Chen IS, Baltimore D. Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5 Proc Natl Acad Sci U S A 2003;100:183-188.
29. Boden D, Pusch O, Lee F, Tucker L, Ramratnam B. Human immunodeficiency virus type 1 escape from RNA interference J Virol 2003;77:11531-11535.
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