- Email: [email protected]
Control of HIV-1 replication by RNA interference
Virus Research 102 (2004) 53–58
Control of HIV-1 replication by RNA interference Nan Sook Lee, John J. Rossi∗ Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA
Abstract Small interfering RNAs (siRNAs) have been shown to direct sequence-specific inhibition of gene expression in mammalian cells. siRNAs are RNA duplexes of 21–23 nucleotides (nts) with ∼2 nt 3 overhangs that can induce degradation of their homologous target mRNAs without interferon responses in mammalian cells. The degradation of the target occurs at the post-transcriptional level, meaning a post-transcriptional gene silencing (PTGS) mechanism called as RNA interference (RNAi). RNAi has emerged as an efficient method to inhibit gene expression in mammalian cells with increasingly successful cases of knockdown of many specific genes. Recent works have shown that the use of RNAi could inhibit HIV-1 replication by targeting viral or cellular genes. RNAi can be considered as a gene-specific therapeutic option for controlling HIV-1 replication. However, the control of HIV-1 replication has become complex because of the limited effectiveness of existing anti-HIV-1 agents and the high speed mutation rate of the HIV-1 genome. Careful assessments are required for the potential of RNAi as a gene therapy approach for controlling HIV-1 replication. This review will discuss the status of the science using RNAi for controlling HIV-1 replication and will describe possible problems for therapeutic applications of RNAi-mediated technologies for HIV-1 behind this novel mechanism. © 2004 Elsevier B.V. All rights reserved. Keywords: RNAi; siRNA; shRNA; HIV-1; PTGS
1. Introduction Gene therapy has gained consideration as a possible treatment for acquired immunodeficiency syndrome (AIDS), either as an alternative or as an addition to anti-retroviral chemotherapy. Several types of RNA gene therapies have been developed and shown to inhibit HIV-1 replication in mammalian cell cultures; these include antisense RNA, catalytic RNA (ribozyme) and high-affinity RNA ligands (aptamers or decoys). Several of these RNA-based approaches have been safety tested in phase I clinical trials, although there are no reports yet of efficacy trials. Nevertheless, in vitro data suggest that their anti-viral efficiencies are variable and can often be overcome by increasing the multiplicity of HIV infectious particles. The limitations have led several investigators to explore the potential of RNA interference (RNAi) as an anti-HIV therapy. RNAi is the process of sequence-specific, post-transcriptional gene silencing (PTGS) in animals and plants triggered by double-stranded RNA (dsRNA) that is homologous in se-
∗
Corresponding author. Tel.: +1-626-301-8360. E-mail address: [email protected] (J.J. Rossi).
0168-1702/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2004.01.015
quence to the silenced gene (Elbashir et al., 2001; Fire et al., 1998; Hutvagner and Zamore, 2002; Sharp, 2001; Zamore et al., 2000). RNAi is initiated by the dsRNA-specific endonuclease Dicer that promotes processive cleavage of long dsRNA into small 21–25 nucleotide (nt) small interfering RNAs (siRNAs) (Bernstein et al., 2001; Hutvagner et al., 2001; Ketting et al., 2001; Knight and Bass, 2001). The duplex siRNA is unwound, and one of the two strands is incorporated into an RNA-induced silencing complex (RISC) (Bernstein et al., 2001; Hammond et al., 2001) guiding it to a homologous target mRNA, which in turn is cleaved by an endonuclease in RISC. This powerful sequence-specific knockdown mechanism has rapidly gained wide acceptance as a surrogate genetic tool and possible therapeutic modality in mammalian cells. It is known that dsRNA of ≥30 bp in size can trigger interferon responses in mammalian cells that are intrinsically sequence-nonspecific to the inducing dsRNA (Paddison et al., 2002b). However, introduction of shorter siRNAs (<30 bp) into mammalian cells leads to mRNA degradation with sequence specificity without activating the interferon response (Elbashir et al., 2001). Synthetic duplexes of 21-nucleotide siRNAs with protruding 3 termini can mediate RNAi in a sequence-specific manner in cultured
54
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mammalian cells. This siRNA technology takes advantage of the fact that RNAi is a natural biological mechanism for silencing genes in most eukaryotic organisms ranging from fission yeast through plants and mammals (Agami, 2002; Cottrell and Doering, 2003; Paddison and Hannon, 2002; Scherr et al., 2003). RNAi functions as an antiviral mechanism in some organisms such as Drosophila and plants, although the role of RNAi in controlling viral infection in mammals is unknown. siRNA-mediated PTGS, therefore, offers a potentially powerful tool for inhibiting HIV replication. This mechanism can be targeted to both viral and cellular transcripts.
2. Targeting HIV-1 with RNAi Introduction of siRNAs specific for HIV-1 into mammalian cells could lead to viral RNA degradation and inhibition of HIV-1 gene expression and replication during different stages of the viral life cycle (Fig. 1). HIV-1 has ∼9 kbp genome that contains nine genes encoding 15 proteins (Greene and Peterlin, 2002). The first potential target for siRNAs is the viral genomic RNA upon viral entry and uncoating. At least one report demonstrates that siRNA-mediated destruction of incoming HIV-1 can take place (Jacque et al., 2002) although other studies of RNAi
Fig. 1. HIV-1 life cycle and potential intervention by RNAi. HIV-1 utilizes the CD4 receptor and the chemokine receptors CCR5/and or CXCR4 for entry. Each of these receptors is a potential RNAi target to block viral entry. In lieu of targeting the receptor or co-receptor mRNAs, incoming genomic viral RNA is a potential target as well, as are all classes of viral transcripts. RISC is the RNAi induced silencing complex that interacts with one of the siRNA strands and positions the strand for complementary base pairing with the target RNA. A component of RISC endonucleolytically cleaves the target RNA allowing the RISC complex to recycle.
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inhibition of retroviral infection suggested that incoming genomic RNA may not be accessible to siRNAs (Hu et al., 2002b; Waterhouse et al., 2001). Once the viral genomic DNA has been integrated, the viral mRNA transcripts as well as the unspliced genomic length RNA are targets for RNAi. Early transcripts such as HIV-1 rev and tat are good targets for siRNAs since the Tat and Rev proteins encoded by these RNAs are essential for subsequent expression of HIV-1 structural genes (Gag, Pol, and Env) and for the synthesis of full length viral genomic RNA. In addition to viral targets, the cellular chemokine receptors CCR5 and CXCR4, which function as co-receptors for HIV-1, have provided new therapeutic targets and a better understanding of the progression of viral infection. CCR5, an HIV-1 co-receptor for M-tropic HIV-1 provides an attractive cellular target for siRNAs since homozygous deletions of CCR5 effectively confer protection from HIV-1 without any serious deleterious effects in immune function (Samson et al., 1996). At least one group has taken advantage of this target for RNAi mediated gene silencing demonstrating in vitro knockdown of CCR5 by siRNAs provided marked protection from HIV-1 infection (Martinez et al., 2002).
3. Exogenous delivery versus endogenous expression of si/shRNAs In mammalian systems, the sequence-specific anti-HIV-1 effects have been observed following introduction of synthetic siRNAs (ssiRNAs) via transfection into HIV-1 infectable cells (Coburn and Cullen, 2002; Gitlin et al., 2002; Jacque et al., 2002; Novina et al., 2002), or via endogenous expression of 21–23 nt transcripts (psiRNAs) or hairpin RNAs (pshRNAs) from DNA plasmids (Jacque et al., 2002;
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Lee et al., 2002a) (Fig. 2). The expression of siRNAs from psiRNAs appears to bypass the need for Dicer or other endonucleases to produce siRNAs (Lee et al., 2002a; Miyagishi and Taira, 2002). The same is true for ssiRNAs, but these must be used in multiple transfections to maintain silencing. The psiRNA system can potentially be used in differentiated mammalian cells containing no or low amounts of Dicer (Kitabwalla and Ruprecht, 2002). However, shRNAs with different stem-loop structures expressed from pshRNAs in mammalian cells require processing to remove the loop. Depending upon the length of the duplex, these may utilize Dicer, but perhaps one or more other endonucleases are involved in this processing step. (Brummelkamp et al., 2002; Parrish et al., 2000; Paul et al., 2002; Sui et al., 2002; Yu et al., 2002; Zeng et al., 2002). The extent of siRNA-directed silencing from both psiRNAs and pshRNAs may be limited by the concentration of active siRNAs in the target cell. The stem-loop structures of shRNAs mimic the naturally occurring stem-loop structures found in micro-RNA precursors which are processed by Dicer to yield small temporal RNAs (stRNA) or microRNA (miRNAs) (Lee et al., 2002b). Processing of anti-HIV siRNAs from a miRNA precursor has recently been demonstrated (Zeng and Cullen, 2002). Inhibition of HIV-1 infection using ssiRNAs, or siRNAs produced from transcription of psiDNAs and pshDNAs has served as a proof-of-principle that siRNA technology can be a possible therapeutic approach for the inhibition of HIV-1 replication in hematopoietic cells (Coburn and Cullen, 2002; Hu et al., 2002a; Jacque et al., 2002; Lee et al., 2002a; Novina et al., 2002). We have demonstrated that a psiRNA approach can be used to inhibit expression of HIV-1 rev and/or tat transcript (s) in both transient transfections (Lee et al., 2002a) or from lentiviral transduced hematopoietic
Fig. 2. Gene expression systems for intracellular transcription of siRNAs or shRNAs. For purposes of simplicity, only the Pol III promoted transcripts are depicted, but Pol II systems can be used as well provided that a polyA signal is included downstream of the shRNA genes. The Pol III transcripts terminate within a string of 5–6 uridines providing a defined 3 end for the transcripts. When sense and antisense strands are transcribed from separate promoters, these strands must anneal to form an siRNA. In contrast, linked shRNA strands readily form a duplex, but the loop joining these must be processed to generate siRNAs.
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progenitor cells (Banerjea et al., 2003). In this approach, the vectors contain two tandem human U6 snRNA promoters followed by 21-mers encoding sense and antisense siRNAs. In the co-transfection experiments, psiRNAs cotransfected with the HIV-1 pNL4-3 proviral DNA led to up to 4 logs of inhibition of HIV-1 p24 antigen expression (Lee et al., 2002a). The high degree of inhibition was achieved by simultaneously targeting two essential sites (rev and tat). It was also demonstrated that synthetic siRNAs targeted to HIV-1 rev and tat mRNAs inhibit HIV-1 gene expression and replication in human T-cell lines and primary lymphocytes (Coburn and Cullen, 2002). Novina et al. (2002) reported that silencing CD4 expression inhibited viral entry, syncytia formation and reduced free viral titers, although CD4 may not be a good therapeutic target because of the receptor’s critical role in T-cell function. Gitlin et al. (2002) reported that siRNA against poliovirus provided intracellular anti-viral immunity to human cells, promoting viral clearance and cell survival. They suggested that gene silencing by siRNAs functions as an adaptive, RNA-based defense mechanism in mammalian cells. Jacque et al. (2002) reported similar results in their studies with HIV-1. They utilized intracellular T7 transcription from a plasmid DNA template to produce siRNAs in the cytoplasm of mammalian cells. Utilizing this system to produce shRNAs targeting HIV-1 vif they were able to confer intracellular immunity to HIV-1 in cell lines as well as primary lymphcytes, in part by blocking reverse transcription of genomic RNA into proviral cDNA. Capodici et al. (2002) used synthetic and in vitro transcribed siRNAs and obtained similar suppression of HIV-1 specific proviral DNA formation and replication in cell lines and primary, activated CD4+ T lymphocytes. In addition to demonstrating that siRNA can inhibit viral infection at pre- and post-integration stages in mammalian cells, they also showed that primary CD4+ T cells could be targeted by siRNAs using lipofectin, or by non-lipid complexed fluorine-derivatized siRNAs. Although transfected siRNAs can effectively inhibit viral replication, the effects are largely transient (Novina et al., 2002). Therefore, even if effective in vivo delivery is obtained, the siRNAs will have to be infused on a regular basis to treat chronic diseases such as AIDS. The use of engineered DNA vectors capable of long term production of siRNAs or shRNAs has facilitated the practical application of RNAi in human primary cells (Brummelkamp et al., 2002; Lee et al., 2002a; Miyagishi and Taira, 2002; Paddison et al., 2002a; Paul et al., 2002; Xia et al., 2002). The demonstrations that RNAi could be activated by expressing siRNAs or shRNAs from eukaryotic promoters has prompted the inclusion of these expression systems into viral vectors that can be used to transduce cells with siRNA or shRNA genes. For treatment of HIV-1 infection, lentiviral vector-based transduction of siRNA and shRNA encoding genes into hematopoietic cells is particularly attractive from the experimental point of view. Lentiviruses are effective for delivery of siRNAs into mammalian cells because they
can effectively transduce both dividing and non-dividing cells and provide long-term gene expression (Abbas-Terki et al., 2002; Matta et al., 2003; Qin et al., 2003; Rubinson et al., 2003; Tiscornia et al., 2003). Anti-CCR5-shRNA genes inserted in a lentiviral vector and introduced into human peripheral blood T lymphocytes (PBLs) resulted in a 10-fold reduction of CCR5 in PBLs and a five-fold reduction of HIV-1 replication. This protection was long term and sequence-specific (Qin et al., 2003). It has also been demonstrated that anti-Rev siRNA genes expressed from lentiviral vector-transduced hematopoietic precursor cells provided in vitro derived macrophages and SCID-hu mouse differentiated T-lymphocytes with long term protection from HIV-1 infection and replication (Banerjea et al., 2003). It should be pointed out that long term RNAi effected by ssiRNAs may also be possible in certain cell types. For instance non-dividing macrophages were shown to be resistant to HIV-1 challenge for over two weeks following treatment with ssiRNAs targeting CCR5(Song et al., 2003). Interestingly, these same investigators demonstrated that the RNAi effect is more long-lived if there is a target RNA present since RNAi was lost in cells that were not continually challenged with viral gene expression.
4. Possible problems for therapeutic applications While the results obtained to date should be considered preliminary in terms of application to humans, they do provide strong justification for further investigating the use of RNAi for treatment of HIV-1 in a clinical setting. It has largely been assumed that siRNAs appear to be under the radar of the host interference response mechanism. It has recently been demonstrated that some expressed shRNAs can activate at least one of the arms of the human interferon response mechanism (Bridge et al., 2003). Since many commonly used tumor cells cannot respond to interferon because of specific mutations of genes in the interferon pathway (Stojdl et al., 2000), careful studies with primary cells need to be carried out to ensure that potentially serious side effects due to interferon induction are not occurring. An additional concern is that the success of RNAi against a viral pathogen is not automatically assured. One important factor is that not all viral mRNA sequences are equally accessible to siRNAs. Upon HIV-1 infection, genomic viral RNA is introduced into the host cell cytoplasm in the form of a nucleoprotein complex that can obscure the recognition by siRNAs. Some viral RNA sequences might be buried within secondary structures or within highly folded regions. It may often be necessary to test several different target sites before a potent RNAi/target combination can be identified. For HIV-1 there is the compounding problem of rapid mutation, rendering the virus resistant to therapeutic agents. Using RNAi agents directed against multiple conserved RNA target sequences in combination with targeting host factors may overcome this problem. Another concern is maintaining
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siRNA expression long term in stably transduced primary cells. It is currently unknown whether or not silencing of Pol III-promoted siRNA and shRNA transcription will be a problem. Finally, since RNAi is a native cellular mechanism that most likely has an important, if not essential function in most cell types, there is the concern that usurping the RNAi machinery with foreign siRNAs could have adverse developmental and functional consequences. Only by carrying out the appropriate experiments in relevant cells and animal models can the potential problems be addressed.
Acknowledgements This work was supported by NIH grants AI 29329, AI42552, and HL074704 to JJR and a GlaxoSmith Kline (GSK) grant to NSL.
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T., Rana, S., Yi, Y., Smyth, R.J., Collman, R.G., Doms, R.W., Vassart, G., Parmentier, M., 1996. Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature 382, 722–725. Scherr, M., Morgan, M.A., Eder, M., 2003. Gene silencing mediated by small interfering RNAs in mammalian cells. Curr. Med. Chem. 10, 245–256. Sharp, P.A., 2001. RNA interference—2001. Genes Dev. 15, 485– 490. Song, E., Lee, S.K., Dykxhoorn, D.M., Novina, C., Zhang, D., Crawford, K., Cerny, J., Sharp, P.A., Lieberman, J., Manjunath, N., Shankar, P., 2003. Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages. J. Virol. 77, 7174–7181. Stojdl, D.F., Lichty, B., Knowles, S., Marius, R., Atkins, H., Sonenberg, N., Bell, J.C., 2000. Exploiting tumor-specific defects in the interferon pathway with a previously unknown oncolytic virus. Nat. Med. 6, 821–825. Sui, G., Soohoo, C., Affar el, B., Gay, F., Shi, Y., Forrester, W.C., 2002. A DNA vector-based RNAi technology to suppress gene ex-
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Control of HIV-1 replication by RNA interference Nan Sook Lee, John J. Rossi∗ Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA
Abstract Small interfering RNAs (siRNAs) have been shown to direct sequence-specific inhibition of gene expression in mammalian cells. siRNAs are RNA duplexes of 21–23 nucleotides (nts) with ∼2 nt 3 overhangs that can induce degradation of their homologous target mRNAs without interferon responses in mammalian cells. The degradation of the target occurs at the post-transcriptional level, meaning a post-transcriptional gene silencing (PTGS) mechanism called as RNA interference (RNAi). RNAi has emerged as an efficient method to inhibit gene expression in mammalian cells with increasingly successful cases of knockdown of many specific genes. Recent works have shown that the use of RNAi could inhibit HIV-1 replication by targeting viral or cellular genes. RNAi can be considered as a gene-specific therapeutic option for controlling HIV-1 replication. However, the control of HIV-1 replication has become complex because of the limited effectiveness of existing anti-HIV-1 agents and the high speed mutation rate of the HIV-1 genome. Careful assessments are required for the potential of RNAi as a gene therapy approach for controlling HIV-1 replication. This review will discuss the status of the science using RNAi for controlling HIV-1 replication and will describe possible problems for therapeutic applications of RNAi-mediated technologies for HIV-1 behind this novel mechanism. © 2004 Elsevier B.V. All rights reserved. Keywords: RNAi; siRNA; shRNA; HIV-1; PTGS
1. Introduction Gene therapy has gained consideration as a possible treatment for acquired immunodeficiency syndrome (AIDS), either as an alternative or as an addition to anti-retroviral chemotherapy. Several types of RNA gene therapies have been developed and shown to inhibit HIV-1 replication in mammalian cell cultures; these include antisense RNA, catalytic RNA (ribozyme) and high-affinity RNA ligands (aptamers or decoys). Several of these RNA-based approaches have been safety tested in phase I clinical trials, although there are no reports yet of efficacy trials. Nevertheless, in vitro data suggest that their anti-viral efficiencies are variable and can often be overcome by increasing the multiplicity of HIV infectious particles. The limitations have led several investigators to explore the potential of RNA interference (RNAi) as an anti-HIV therapy. RNAi is the process of sequence-specific, post-transcriptional gene silencing (PTGS) in animals and plants triggered by double-stranded RNA (dsRNA) that is homologous in se-
∗
Corresponding author. Tel.: +1-626-301-8360. E-mail address: [email protected] (J.J. Rossi).
0168-1702/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2004.01.015
quence to the silenced gene (Elbashir et al., 2001; Fire et al., 1998; Hutvagner and Zamore, 2002; Sharp, 2001; Zamore et al., 2000). RNAi is initiated by the dsRNA-specific endonuclease Dicer that promotes processive cleavage of long dsRNA into small 21–25 nucleotide (nt) small interfering RNAs (siRNAs) (Bernstein et al., 2001; Hutvagner et al., 2001; Ketting et al., 2001; Knight and Bass, 2001). The duplex siRNA is unwound, and one of the two strands is incorporated into an RNA-induced silencing complex (RISC) (Bernstein et al., 2001; Hammond et al., 2001) guiding it to a homologous target mRNA, which in turn is cleaved by an endonuclease in RISC. This powerful sequence-specific knockdown mechanism has rapidly gained wide acceptance as a surrogate genetic tool and possible therapeutic modality in mammalian cells. It is known that dsRNA of ≥30 bp in size can trigger interferon responses in mammalian cells that are intrinsically sequence-nonspecific to the inducing dsRNA (Paddison et al., 2002b). However, introduction of shorter siRNAs (<30 bp) into mammalian cells leads to mRNA degradation with sequence specificity without activating the interferon response (Elbashir et al., 2001). Synthetic duplexes of 21-nucleotide siRNAs with protruding 3 termini can mediate RNAi in a sequence-specific manner in cultured
54
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mammalian cells. This siRNA technology takes advantage of the fact that RNAi is a natural biological mechanism for silencing genes in most eukaryotic organisms ranging from fission yeast through plants and mammals (Agami, 2002; Cottrell and Doering, 2003; Paddison and Hannon, 2002; Scherr et al., 2003). RNAi functions as an antiviral mechanism in some organisms such as Drosophila and plants, although the role of RNAi in controlling viral infection in mammals is unknown. siRNA-mediated PTGS, therefore, offers a potentially powerful tool for inhibiting HIV replication. This mechanism can be targeted to both viral and cellular transcripts.
2. Targeting HIV-1 with RNAi Introduction of siRNAs specific for HIV-1 into mammalian cells could lead to viral RNA degradation and inhibition of HIV-1 gene expression and replication during different stages of the viral life cycle (Fig. 1). HIV-1 has ∼9 kbp genome that contains nine genes encoding 15 proteins (Greene and Peterlin, 2002). The first potential target for siRNAs is the viral genomic RNA upon viral entry and uncoating. At least one report demonstrates that siRNA-mediated destruction of incoming HIV-1 can take place (Jacque et al., 2002) although other studies of RNAi
Fig. 1. HIV-1 life cycle and potential intervention by RNAi. HIV-1 utilizes the CD4 receptor and the chemokine receptors CCR5/and or CXCR4 for entry. Each of these receptors is a potential RNAi target to block viral entry. In lieu of targeting the receptor or co-receptor mRNAs, incoming genomic viral RNA is a potential target as well, as are all classes of viral transcripts. RISC is the RNAi induced silencing complex that interacts with one of the siRNA strands and positions the strand for complementary base pairing with the target RNA. A component of RISC endonucleolytically cleaves the target RNA allowing the RISC complex to recycle.
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inhibition of retroviral infection suggested that incoming genomic RNA may not be accessible to siRNAs (Hu et al., 2002b; Waterhouse et al., 2001). Once the viral genomic DNA has been integrated, the viral mRNA transcripts as well as the unspliced genomic length RNA are targets for RNAi. Early transcripts such as HIV-1 rev and tat are good targets for siRNAs since the Tat and Rev proteins encoded by these RNAs are essential for subsequent expression of HIV-1 structural genes (Gag, Pol, and Env) and for the synthesis of full length viral genomic RNA. In addition to viral targets, the cellular chemokine receptors CCR5 and CXCR4, which function as co-receptors for HIV-1, have provided new therapeutic targets and a better understanding of the progression of viral infection. CCR5, an HIV-1 co-receptor for M-tropic HIV-1 provides an attractive cellular target for siRNAs since homozygous deletions of CCR5 effectively confer protection from HIV-1 without any serious deleterious effects in immune function (Samson et al., 1996). At least one group has taken advantage of this target for RNAi mediated gene silencing demonstrating in vitro knockdown of CCR5 by siRNAs provided marked protection from HIV-1 infection (Martinez et al., 2002).
3. Exogenous delivery versus endogenous expression of si/shRNAs In mammalian systems, the sequence-specific anti-HIV-1 effects have been observed following introduction of synthetic siRNAs (ssiRNAs) via transfection into HIV-1 infectable cells (Coburn and Cullen, 2002; Gitlin et al., 2002; Jacque et al., 2002; Novina et al., 2002), or via endogenous expression of 21–23 nt transcripts (psiRNAs) or hairpin RNAs (pshRNAs) from DNA plasmids (Jacque et al., 2002;
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Lee et al., 2002a) (Fig. 2). The expression of siRNAs from psiRNAs appears to bypass the need for Dicer or other endonucleases to produce siRNAs (Lee et al., 2002a; Miyagishi and Taira, 2002). The same is true for ssiRNAs, but these must be used in multiple transfections to maintain silencing. The psiRNA system can potentially be used in differentiated mammalian cells containing no or low amounts of Dicer (Kitabwalla and Ruprecht, 2002). However, shRNAs with different stem-loop structures expressed from pshRNAs in mammalian cells require processing to remove the loop. Depending upon the length of the duplex, these may utilize Dicer, but perhaps one or more other endonucleases are involved in this processing step. (Brummelkamp et al., 2002; Parrish et al., 2000; Paul et al., 2002; Sui et al., 2002; Yu et al., 2002; Zeng et al., 2002). The extent of siRNA-directed silencing from both psiRNAs and pshRNAs may be limited by the concentration of active siRNAs in the target cell. The stem-loop structures of shRNAs mimic the naturally occurring stem-loop structures found in micro-RNA precursors which are processed by Dicer to yield small temporal RNAs (stRNA) or microRNA (miRNAs) (Lee et al., 2002b). Processing of anti-HIV siRNAs from a miRNA precursor has recently been demonstrated (Zeng and Cullen, 2002). Inhibition of HIV-1 infection using ssiRNAs, or siRNAs produced from transcription of psiDNAs and pshDNAs has served as a proof-of-principle that siRNA technology can be a possible therapeutic approach for the inhibition of HIV-1 replication in hematopoietic cells (Coburn and Cullen, 2002; Hu et al., 2002a; Jacque et al., 2002; Lee et al., 2002a; Novina et al., 2002). We have demonstrated that a psiRNA approach can be used to inhibit expression of HIV-1 rev and/or tat transcript (s) in both transient transfections (Lee et al., 2002a) or from lentiviral transduced hematopoietic
Fig. 2. Gene expression systems for intracellular transcription of siRNAs or shRNAs. For purposes of simplicity, only the Pol III promoted transcripts are depicted, but Pol II systems can be used as well provided that a polyA signal is included downstream of the shRNA genes. The Pol III transcripts terminate within a string of 5–6 uridines providing a defined 3 end for the transcripts. When sense and antisense strands are transcribed from separate promoters, these strands must anneal to form an siRNA. In contrast, linked shRNA strands readily form a duplex, but the loop joining these must be processed to generate siRNAs.
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progenitor cells (Banerjea et al., 2003). In this approach, the vectors contain two tandem human U6 snRNA promoters followed by 21-mers encoding sense and antisense siRNAs. In the co-transfection experiments, psiRNAs cotransfected with the HIV-1 pNL4-3 proviral DNA led to up to 4 logs of inhibition of HIV-1 p24 antigen expression (Lee et al., 2002a). The high degree of inhibition was achieved by simultaneously targeting two essential sites (rev and tat). It was also demonstrated that synthetic siRNAs targeted to HIV-1 rev and tat mRNAs inhibit HIV-1 gene expression and replication in human T-cell lines and primary lymphocytes (Coburn and Cullen, 2002). Novina et al. (2002) reported that silencing CD4 expression inhibited viral entry, syncytia formation and reduced free viral titers, although CD4 may not be a good therapeutic target because of the receptor’s critical role in T-cell function. Gitlin et al. (2002) reported that siRNA against poliovirus provided intracellular anti-viral immunity to human cells, promoting viral clearance and cell survival. They suggested that gene silencing by siRNAs functions as an adaptive, RNA-based defense mechanism in mammalian cells. Jacque et al. (2002) reported similar results in their studies with HIV-1. They utilized intracellular T7 transcription from a plasmid DNA template to produce siRNAs in the cytoplasm of mammalian cells. Utilizing this system to produce shRNAs targeting HIV-1 vif they were able to confer intracellular immunity to HIV-1 in cell lines as well as primary lymphcytes, in part by blocking reverse transcription of genomic RNA into proviral cDNA. Capodici et al. (2002) used synthetic and in vitro transcribed siRNAs and obtained similar suppression of HIV-1 specific proviral DNA formation and replication in cell lines and primary, activated CD4+ T lymphocytes. In addition to demonstrating that siRNA can inhibit viral infection at pre- and post-integration stages in mammalian cells, they also showed that primary CD4+ T cells could be targeted by siRNAs using lipofectin, or by non-lipid complexed fluorine-derivatized siRNAs. Although transfected siRNAs can effectively inhibit viral replication, the effects are largely transient (Novina et al., 2002). Therefore, even if effective in vivo delivery is obtained, the siRNAs will have to be infused on a regular basis to treat chronic diseases such as AIDS. The use of engineered DNA vectors capable of long term production of siRNAs or shRNAs has facilitated the practical application of RNAi in human primary cells (Brummelkamp et al., 2002; Lee et al., 2002a; Miyagishi and Taira, 2002; Paddison et al., 2002a; Paul et al., 2002; Xia et al., 2002). The demonstrations that RNAi could be activated by expressing siRNAs or shRNAs from eukaryotic promoters has prompted the inclusion of these expression systems into viral vectors that can be used to transduce cells with siRNA or shRNA genes. For treatment of HIV-1 infection, lentiviral vector-based transduction of siRNA and shRNA encoding genes into hematopoietic cells is particularly attractive from the experimental point of view. Lentiviruses are effective for delivery of siRNAs into mammalian cells because they
can effectively transduce both dividing and non-dividing cells and provide long-term gene expression (Abbas-Terki et al., 2002; Matta et al., 2003; Qin et al., 2003; Rubinson et al., 2003; Tiscornia et al., 2003). Anti-CCR5-shRNA genes inserted in a lentiviral vector and introduced into human peripheral blood T lymphocytes (PBLs) resulted in a 10-fold reduction of CCR5 in PBLs and a five-fold reduction of HIV-1 replication. This protection was long term and sequence-specific (Qin et al., 2003). It has also been demonstrated that anti-Rev siRNA genes expressed from lentiviral vector-transduced hematopoietic precursor cells provided in vitro derived macrophages and SCID-hu mouse differentiated T-lymphocytes with long term protection from HIV-1 infection and replication (Banerjea et al., 2003). It should be pointed out that long term RNAi effected by ssiRNAs may also be possible in certain cell types. For instance non-dividing macrophages were shown to be resistant to HIV-1 challenge for over two weeks following treatment with ssiRNAs targeting CCR5(Song et al., 2003). Interestingly, these same investigators demonstrated that the RNAi effect is more long-lived if there is a target RNA present since RNAi was lost in cells that were not continually challenged with viral gene expression.
4. Possible problems for therapeutic applications While the results obtained to date should be considered preliminary in terms of application to humans, they do provide strong justification for further investigating the use of RNAi for treatment of HIV-1 in a clinical setting. It has largely been assumed that siRNAs appear to be under the radar of the host interference response mechanism. It has recently been demonstrated that some expressed shRNAs can activate at least one of the arms of the human interferon response mechanism (Bridge et al., 2003). Since many commonly used tumor cells cannot respond to interferon because of specific mutations of genes in the interferon pathway (Stojdl et al., 2000), careful studies with primary cells need to be carried out to ensure that potentially serious side effects due to interferon induction are not occurring. An additional concern is that the success of RNAi against a viral pathogen is not automatically assured. One important factor is that not all viral mRNA sequences are equally accessible to siRNAs. Upon HIV-1 infection, genomic viral RNA is introduced into the host cell cytoplasm in the form of a nucleoprotein complex that can obscure the recognition by siRNAs. Some viral RNA sequences might be buried within secondary structures or within highly folded regions. It may often be necessary to test several different target sites before a potent RNAi/target combination can be identified. For HIV-1 there is the compounding problem of rapid mutation, rendering the virus resistant to therapeutic agents. Using RNAi agents directed against multiple conserved RNA target sequences in combination with targeting host factors may overcome this problem. Another concern is maintaining
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siRNA expression long term in stably transduced primary cells. It is currently unknown whether or not silencing of Pol III-promoted siRNA and shRNA transcription will be a problem. Finally, since RNAi is a native cellular mechanism that most likely has an important, if not essential function in most cell types, there is the concern that usurping the RNAi machinery with foreign siRNAs could have adverse developmental and functional consequences. Only by carrying out the appropriate experiments in relevant cells and animal models can the potential problems be addressed.
Acknowledgements This work was supported by NIH grants AI 29329, AI42552, and HL074704 to JJR and a GlaxoSmith Kline (GSK) grant to NSL.
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