Realizing the Promise of RNAi
Authors: John J Rossi
Journal: Molecular Therapy (2008)

The convergence of the discovery of RNA interference (RNAi) with the completion of the human genome sequencing program has revolutionized our methods of analyzing gene function in mammalian cells.1Huppi K Martin SE Caplen NJ Defining and assaying RNAi in mammalian cells.Mol Cell. 2005; 17: 1-10Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar,2Silva J Chang K Hannon GJ Rivas FV RNA-interference-based functional genomics in mammalian cells: reverse genetics coming of age.Oncogene. 2004; 23: 8401-8409Crossref PubMed Scopus (100) Google Scholar The task of studying gene function can be relatively easily accomplished by the use of small interfering RNAs (siRNAs) for targeted inhibition of gene expression. Furthermore, use of RNAi library screens to identify specified phenotypes can be applied to drug target development.3Paddison PJ RNA interference in mammalian cell systems.Curr Topics Microbiol Immunol. 2008; 320: 1-19PubMed Google Scholar As such, a recent publication in Science by Brass et al.4Brass AL Dykxhoorn DM Benita Y Yan N Engelman A Xavier RJ et al.Identification of host proteins required for HIV infection through a functional genomic screen.Science. 2008; 319: 921-926Crossref PubMed Scopus (1183) Google Scholar has stimulated a great deal of interest in the AIDS research community. This group, led by Stephen Elledge and Judith Lieberman, published an exciting application of RNAi that used an siRNA library screen to identify cellular factors required for HIV entry, replication, and infectious virus production. A handful of essential cellular host factors had previously been determined by other techniques, but this latest screen revealed more than 250 new cellular proteins required for HIV-1 to complete its replication cycle. The team termed these genes host dependency factors (HDFs). It is exciting to speculate that small-molecule drugs may be developed for some of these HDF targets in the next few years and that there may even be existing drugs that inhibit some of these HDFs that can now be developed for treating HIV-1 infection. RNAi is a mechanism in which small double-stranded RNA molecules of 21–25 bases in length are incorporated into the RNA-induced silencing complex and effect post-transcriptional and transcriptional gene silencing.5Hannon GJ Rossi JJ Unlocking the potential of the human genome with RNA interference.Nature. 2004; 431: 371-378Crossref PubMed Scopus (973) Google Scholar,6Hannon GJ RNA interference.Nature. 2002; 418: 244-251Crossref PubMed Scopus (3543) Google Scholar In post-transcriptional gene silencing, the small RNAs serve as guides for sequence-specific silencing of gene expression.7Scherer LJ Rossi JJ Approaches for the sequence-specific knockdown of mRNA.Nat Biotechnol. 2003; 21: 1457-1465Crossref PubMed Scopus (407) Google Scholar Any sequence is a potential target for RNAi, in that Watson–Crick base-pairing of an RNAi guide strand with a targeted message will effect sequence-specific degradation of the target. When siRNAs are used to trigger targeted degradation of a messenger RNA, RNAi is efficient enough to obtain greater than 90% inhibition of target transcript expression in many instances. The use of RNAi as a tool for studying gene function is nicely complemented by the potential application of the same siRNAs as therapeutic agents.5Hannon GJ Rossi JJ Unlocking the potential of the human genome with RNA interference.Nature. 2004; 431: 371-378Crossref PubMed Scopus (973) Google Scholar,8Behlke MA Progress towards in vivo use of siRNAs.Mol Ther. 2006; 13: 644-670Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar Thus far, much work has already been carried out using RNAi to understand basic cellular processes for cancer and metabolic diseases, and siRNAs are in clinical trials for treatment of both metabolic and viral diseases. Libraries of siRNAs or expressed short hairpin RNAs (shRNAs), which are processed into siRNAs, are available from both academic and commercial sources. The primary use of these libraries has been to identify targets for small-drug or RNAi therapy. Finding druggable targets has and will continue to be an important part of new drug development. The treatment of HIV infection poses a significant problem for antiretroviral drug development, because the virus rapidly evolves resistance to drugs targeting viral proteins or nucleic acids. For HIV-1-infected individuals with multiple drug-resistant viruses, a potential solution for therapy is the use of drugs that safely target cellular factors required for HIV-1 infection.9Lunzen J Treatment of heavily antiretroviral-experienced HIV-infected patients.AIDS Rev. 2007; 9: 246-253PubMed Google Scholar The discovery of the nonessential chemokine receptor CCR5 as a cellular co-factor used by HIV for infection10Samson M Libert F Doranz BJ Rucker J Liesnard C Farber CM et al.Resistance to HIV-1 infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene.Nature. 1996; 382: 722-725Crossref PubMed Scopus (2473) Google Scholar raised great hopes that cellular targets could be used for treatment of HIV infection. In fact, there are now clinically approved drugs that block the use of this co-receptor, but not all patients can take these compounds.11van Lunzen J How will CCR5 antagonists influence the recommendations for the antiretroviral treatment of HIV-1 infection?.Eur J Med Res. 2007; 12: 435-440PubMed Google Scholar Aside from this discovery more than 10 years ago, there have been no newly discovered cellular factors that are good candidates for anti-HIV drug development. In the new study by Brass et al., the investigators took advantage of the fact that HeLa cells express the HIV-1 co-receptor CXCR4 and can be engineered to co-express the major HIV-1 receptor CD4 as well as the other co-receptor CCR5. Therefore, engineered HeLa cells are readily infected by HIV-1. Moreover, HeLa cells are easy to transfect with siRNAs in complex with lipids, making them amenable to an siRNA library screen. Twenty-four hours after transfecting the engineered HeLa cells with the siRNA library members, Brass and colleagues subjected the cells to an acute challenge with HIV-1 IIIB. The siRNA library targeted more than 21,000 cellular genes and used four siRNAs per gene. The investigators used two strategies to identify cellular targets required for early vs. late stages of viral replication and assembly. The first strategy used reduction in HIV p24 levels to determine the HDFs required for all steps in the viral life cycle before mature virus production. The second assay monitored the production of infectious viral particles using an HIV TAT-inducible reporter cell line that can be challenged with HIV for quantification of infectious viral particles. The combined screens identified 273 HDFs, 237 of which constitute novel HDFs for HIV-1. More than 100 of the 237 new HDFs had two or more of the siRNAs targeting them, providing a high level of confidence in the validity of the screen results. The cellular factors identified in this screen revealed both expected and unexpected candidates. Reassuringly, the screen identified 36 cellular host factors previously shown to be required for HIV-1 replication. HDFs that are part of essential cellular pathways required for HIV infection, such as components of the nuclear pore complex and transcription-associated factors, were also identified. Twenty-eight of the HDFs were exclusive to the second screening procedure, pointing to a pivotal role for these HDFs in viral assembly and egress. Among the unexpected HDFs was an assembly of proteins involved in autophagy, a process used to degrade and recycle cellular components. One such component, Rab6A, regulates retrograde transport from Golgi to endoplasmic reticulum. Selective knockdown of this protein revealed that it is required for an early step in the infection process, before reverse transcription. Moreover, knockdown of Rab6A had no effect on a vesicular stomatitis virus G pseudo-typed version of HIV or a retroviral vector. Thus, the authors concluded that HIV-1 fusion to cellular membranes during early stages of infection requires retrograde transport of vesicles from the endosome to the Golgi. Other classes of proteins that were identified as HDFs for HIV-1 include helicases, hydrolases, actin-binding proteins, DNA-directed RNA polymerase–associated proteins, dolichoyl-diphospho-oligosaccharide protein glycol transferases, guanosine triphosphate and enzyme-binding proteins, and, intriguingly, RNA methyl transferases. Overall, there were 17 classes of HDFs whose loss significantly affected HIV-1 replication. Because these analyses were carried out in HeLa cells, the authors asked if any of the HDFs were expressed at high levels in lymphocytes, the natural host for HIV-1. They found that a significant percentage of the HDFs are expressed at elevated levels in lymphocytes, thus contributing to the role of these cells as a preferred target for HIV-1 infection and replication. HIV-1 is a relatively simple virus, encoding only a handful of its own proteins. Perhaps this explains why it has evolved such a large dependency on host factors for its infectious cycle. Studies of viruses most often lead to an increased knowledge of host cellular functions. This latest study will provide many years of new research opportunities to study the relationship between HIV-1 and the HDFs it requires, and will also lead to a greater understanding of the functional roles many of these HDFs play in normal cellular physiology. To some, one of the more exciting aspects of this work is the idea that siRNAs that target these HDFs could also become drugs. There is concern, however, that targeting HDFs with siRNAs could lead to toxicity, given that these factors are needed for normal cellular function. This potential problem may be addressed by selective siRNA delivery or shRNA expression schemes. As an example, Judy Lieberman and colleagues have already demonstrated that a chimeric antibody fragment–protamine fusion targeting the HIV envelope can selectively deliver siRNAs to HIV-infected T cells.12Song E Zhu P Lee SK Chowdhury D Kussman S Dykxhoorn DM et al.Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors.Nat Biotechnol. 2005; 23: 709-717Crossref PubMed Scopus (915) Google Scholar Perhaps this approach could be used for systemic, targeted delivery of anti-HDF siRNAs in patients. Alternatively, if a gene therapy approach is considered, the use of a TAT-inducible shRNA expression system such as that described by Unwalla et al. would minimize toxicity by allowing the shRNAs to function only in HIV-infected cells.13Unwalla HJ Li MJ Kim JD Li HT Ehsani A Alluin J et al.Negative feedback inhibition of HIV-1 by TAT-inducible expression of siRNA.Nat Biotechnol. 2004; 22: 1573-1578Crossref PubMed Scopus (74) Google Scholar Thus, the use of RNAi as a tool for better understanding the host factors required for HIV-1 infection may rapidly lead to new therapeutic targets and agents for the treatment of HIV. Ten years ago, when Fire and Mello discovered RNAi,14Fire A Xu S Montgomery MK Kostas SA Driver SE Mello CC Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature. 1998; 391: 806-811Crossref PubMed Scopus (11906) Google Scholar there was speculation that this cellular mechanism could be a valuable tool for elucidating gene function. It is amazing how rapidly this powerful mechanism would lead to new discoveries—and even new therapies.