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fistulizing disease also had higher infliximab concentrations than those without fistulizing disease in each patient stratum. Perhaps the most important finding from the study by Baert et al. (N Engl J Med 2003;348:601– 608) is that concomitant immunosuppressive therapy reduces the immunogenicity of infliximab. This observation led to the recommendations by the authors that concurrent immunomodulators should be used with infliximab therapy. Clearly, treatment with episodic infliximab infusions without concurrent immunosuppressive therapy is a relatively immunogenic regimen and should be avoided. The important question facing clinicians is whether concomitant immunosuppressive therapy has the most favorable benefit versus risk profile in terms of efficacy, immunogenicity, and its associated implications, and potential toxicities of polypharmacy. In the ACCENT I study, the rates of antibodies to infliximab were 38%, 11%, and 8%, respectively, in patients without concomitant immunomodulators receiving episodic infusions, 5 mg/ kg, or 10 mg/kg maintenance therapy, compared with 16%, 7%, and 4% in patients receiving concomitant immunomodulators in the same respective infliximab dosage groups. The difference in the incidence of antibody formation between patients with and without concomitant immunomodulators was statistically significant in episodically treated patients, but not those who received maintenance therapy (personal communication, Thomas Schaible, Ph.D., Centocor, Malvern, PA, August 2003). Because the use of concurrent immunomodulatory agents has not been shown to result in a statistically significant additional benefit in patients receiving infliximab maintenance therapy, and immunomodulatory therapy is associated with potential toxicities, it is unclear whether infliximab maintenance therapy alone or in conjunction with concomitant immunomodulators is the most optimal regimen. As we learn more about the impact of immunomodulator therapy on immunogenicity of infliximab and whether it should be recommended to be concurrently administered with infliximab therapy, many questions remain to be answered. The overall incidence of antibody formation was low in patients with rheumatoid arthritis receiving infliximab maintenance therapy in combination with methotrexate, which is probably not as widely used as azathioprine or 6-mercaptopurine in Crohn’s disease. It is unclear whether different immunosuppressive agents offer varying degrees of protection against the development of antibodies against infliximab. It is equally uncertain how they should be used to minimize immunogenicity, if at all. In the ACCENT I study, the overall incidence of antibodies to infliximab was 16.9% in patients on concurrent steroids alone compared with 10% in patients on concurrent immunomodulators alone (Gastroenterology 2002;122:A613). A recent study reported that intravenous hydrocortisone premedication prior to infliximab infusions reduces the concentrations of antibodies against infliximab, although such a strategy does not result in a statistically significant reduction in the incidence of antibody formation (Gastroenterology 2003;124:917–924). Furthermore, based on the data presented by Baert et al. (N Engl J Med 2003;348:601– 608), the protective benefit of concomitant immunomodulators may have a window period and may require their initiation prior to the first infliximab infusion. Given the delayed onset of action for these medications, particularly azathioprine and 6-mercaptopurine, the precise timing, dosing, and duration of concurrent immunomodulatory therapy in conjunction with infliximab therapy poses uncertainty to clinicians who are often faced with the challenges of providing rapid relief for the patient’s symptoms while minimizing the number of prescribed medications.

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Can the immunogenicity be eliminated with humanized or fully human therapeutic monoclonal antibodies? Unfortunately, the formation of antibodies is not a problem unique to infliximab, nor is it necessarily a function of infliximab’s being a chimeric molecule. It is well known that the administration of any protein, including those that are fully human, may be associated with the development of antibodies. For example, 44% of patients develop antibodies to recombinant human insulin (Diabetologia 1983;25:465– 469), whereas 24% of patients develop antibodies against recombinant human factor VIII (Hemophilia 1998;4:552–557). It is particularly notable that up to 12% of patients develop antibodies against the recently developed, fully human anti–tumor necrosis factor monoclonal antibody adalimumab during scheduled maintenance therapy with this drug (Humira, US label, 2003). Antibodies also develop in up to 7% of patients receiving a different type of therapeutic molecule, the recombinant humanized antibody against ␣4 integrin, natalizumab (Elan Pharmaceuticals and Biogen) (N Engl J Med 2003;348:24 –32). The mechanisms underlying a protein’s immunogenicity are complex and poorly understood, and go far beyond the species from which a therapeutic protein is derived. It is likely that immunogenicity will be an issue for most, if not all, therapeutic proteins. To minimize this problem, regularly scheduled maintenance therapy with the avoidance of long drug holidays is likely the preferred treatment strategy for all protein biopharmaceuticals that must be given on a repeated basis. The overall goal of treatment in Crohn’s disease is to keep patients in remission and off corticosteroids with an agent that is relatively safe and whose benefit persists over the long term. Infliximab has demonstrated the ability to bring about these benefits, but as this study highlights, the development of antibodies to infliximab may mute this effect. It is clear that episodic treatment with infliximab in the absence of concurrent immunosuppressive therapy is not advisable. It is less likely, but needs formal confirmation, that regularly scheduled maintenance therapy alone is the optimal approach for using infliximab in the treatment of Crohn’s disease. Future studies, including randomized-controlled trials, are necessary to clarify the need for concomitant immunomodulatory therapy and its dosing regimen in infliximab therapy for patients with Crohn’s disease. CHINYU G. SU, M.D. GARY R. LICHTENSTEIN, M.D.

INCREASING THE RISC FOR HCV McCaffrey AP, Meuse L, Pham TT, Conklin DS, Hannon GJ, Kay MA (Program in Human Gene Therapy, Departments of Pediatrics and Genetics, Stanford University School of Medicine, Stanford, California; Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). RNA interference in adult mice. Nature 2002;418:38 –39. RNA interference (RNAi) is a highly conserved, post-transcriptional, homology-dependent gene silencing mechanism used by eukaryotic cells to target destruction of messenger RNA (mRNA) to control gene expression during development and combat threats to genome integrity, such as viral infection and transposon mobilization (Nature 2002;418:244 –251). First recognized and most extensively studied in the nematode worm Caenorhabditis elegans and in plants, RNAi has been identified in a wide array of organisms including flies, fungi,

November 2003

protozoa, and, recently, mammals (Nature 2001;411:494 – 498, Cell 1995;81:611– 620, Plant Cell 1990;2:279 –289, Nature 1998;391:806 – 811). RNAi is initiated by small interfering RNA (siRNA), a double stranded form of RNA 21-23 bases in length, usually generated by cleavage of larger doublestranded transcripts by an endonuclease complex aptly called Dicer (Nat Rev Genet 2002;3:737–747, Genes Dev 2001;15: 188 –200). Experimentally, RNAi can also be accomplished by introducing synthetic siRNA directly into cells by transfection or expressing siRNA precursors (small hairpin RNAs) from DNA templates (Science 2002;296:550 –553). siRNAs introduced into cells by either route assemble with a multi-protein complex termed RNA-inducing silencing complex (RISC) that uses the siRNA as a guide to identify and degrade homologous mRNA target sequence, thus acting as a sequence-specific nuclease. The brief report by McCaffrey et al. (Nature 2002;418:38 – 39) is noteworthy because it was the first to demonstrate RNAi in vivo in adult mammals. In a series of experiments, the investigators use RNAi to target expression of a plasmid encoding a chimeric HCV-luciferase fusion protein as a single open reading frame. The HCV sequences contained in the plasmid represent the viral polymerase protein, NS5B (nonstructural protein 5B). Expression of both the chimeric plasmid and siRNAs targeting sequences for either luciferase or HCV in mouse liver was achieved using hydrodynamic transfection, a technique that results in relatively high-level but transient hepatic expression (Gene Ther 1999;6:1258 –1266, Hum Gene Ther 1999;10:1735–1737). The in vivo expression of luciferase was subsequently assessed by surface scanning with a ultra-sensitive charge coupled device (CCD) camera (Annu Rev Biomed Eng 2002;4:235–260). The investigators found that coexpression of the chimeric plasmid with either antiluciferase or anti-NS5B siRNAs, whether chemically synthesized or transcribed in vivo from plasmids, reduced luciferase expression by approximately 80%–90% when compared with control siRNAs. Comment. Approximately 2% of the world’s population is chronically infected with hepatitis C virus (HCV), and complications of chronic infection, including decompensated cirrhosis and hepatoma, represent enormous public health problems. Effective vaccines for HCV have been elusive, and current therapy is expensive, cumbersome, and unsuccessful in most patients. Clearly new and improved therapies are needed. This short report by McCaffrey et al. describes the application of RNAi, one of the most exciting and rapidly moving areas in biology, to the problem of hepatitis C. RNAi has rapidly become the method of choice to knock down expression of genes in mammalian cells largely because of its impressive specificity; even a single base mismatch between an siRNA and its mRNA target dramatically decreases gene silencing. In contrast, conventional antisense technology has been plagued by poor specificity because the introduction of double-stranded RNA of greater than 30 base pairs in length into mammalian cells triggers the interferon pathway, resulting in nonspecific RNA degradation and global inhibition of protein translation (Nat Rev Genet 2002;3:737–747). Furthermore, siRNAs are generally much easier to design and produce than ribozymes. As a result of its specificity and ease of use, RNAi is

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already being widely used to develop genetic screens for therapeutic targets, to produce transgenic “knock down” mice, and for large-scale functional genomics (Nature 2002;418:244 –251). This report by McCaffrey et al., the first to demonstrate RNAi in vivo in adult mammals, was quickly followed by 2 others, both also using hydrodynamic transfection of siRNAs into mouse liver to initiate RNAi. The first, from Lewis et al, confirmed that cotransfection of a plasmid expressing firefly luciferase with an anti-luciferase siRNA resulted in significant (80%–90%), specific, and dose-dependent inhibition of luciferase gene expression (Nat Genet 2002;32: 107–108). In additional experiments, the investigators knocked down expression of a secreted form of human placental alkaline phosphatase, and also demonstrated that RNAi could be used to inhibit a transgene (for green fluorescence protein) expressed from the genome of transgenic mice, although they did not quantify the degree of inhibition. In the second report, Song et al. used RNAi to knock down expression of an endogenous gene (as opposed to a transgene) with meaningful biological effects (Nat Med 2003;9:347–351). In a series of experiments, they showed that siRNAs directed against the Fas death receptor protect mice against an otherwise lethal challenge with either an apoptosis-inducing anti-Fas antibody or concanavalin A. Like in the study by McCaffery, hydrodynamic transfection was used to deliver the siRNAs to mouse liver, and in this case almost 90% of hepatocytes seemed to be transfected. In mice pretreated with the anti-fas siRNA, the severe liver injury usually associated with administration of anti-fas antibody or concanavalin A was markedly abrogated and survival was improved. Furthermore, treatment with the anti-Fas siRNA appeared to be beneficial even after the initiation of liver injury and prevented the histological cirrhosis that was otherwise associated with repetitive concanavalin A administration (Nat Med 2003;9:347–351). As these reports suggest, RNAi has a number of exciting potential applications. Relevant to the report by McCaffrey et al., the application of RNAi to combat viral infection seems a natural one because RNAi is thought to have evolved, at least in some organisms, as an antiviral defense. Not surprisingly, therefore, a number of different viral infections, including HCV, HIV, HBV, influenza, polio, and others, have been among the earliest targets of therapeutic RNAi (Proc Natl Acad Sci U S A 2003;100:2718 –2723, Proc Natl Acad Sci U S A 2003;100:2014 –2018, Nat Med 2002;8:681– 686, Proc Natl Acad Sci U S A 2003;100:183–188, Proc Natl Acad Sci U S A 2003;100:235–240, Hepatology 2003;37:764 –770, Nature 2002; 418:430 – 434). However, recent work using RNAi against the polio virus has demonstrated the sobering but not unanticipated finding that mutation of target sequences in the viral genome occur rapidly and may limit the therapeutic potential of RNAi for viral infections (Nature 2002;418:430 – 434). Given the notorious propensity of HCV to mutate, it is likely that this will represent a major practical barrier to the application of RNAi against chronic HCV infection. Here, a major attribute of RNAi, its exquisite specificity, is a liability because even a single base pair mutation can completely abolish RNAi. Several strategies might be used to circumvent this problem. For example, targeting conserved areas or several simultaneous sites of the HCV genome should theoretically decrease the likelihood of selecting for HCV escape mutants. However, there is no guarantee that conserved areas, such as the 5⬘ region of the HCV genome, will be accessible to the RNAi machinery because they are often highly structured and protein-bound, and may thus be largely inaccessible to RISC. Targeting several sites of the HCV genome simultaneously may also be problematic if the RNAi machinery is limiting; in this situation, targeting 2 or more sites may be no better or even worse

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than targeting just one. Perhaps the most promising strategy to combat viruses like HCV with RNAi will involve targeting the destruction of cell constituents that are essential to viral pathogenesis but not to cell survival, as this approach should be less influenced by viral mutation (Nat Med 2002;8:681– 686). Combined with the ability to stably generate siRNAs from plasmid or other vectors, such an approach may effectively allow for intracellular immunization against specific viral infections (Nat Rev Genet 2002;3:737–747). Furthermore, if HCV-resistant hepatocytes can be generated by such an approach, they may have a selective survival advantage over HCV-infected cells and may thereby repopulate the liver with healthy, virus-resistant cells. Like viral disease, use of RNAi for cancer therapy has gained wide attention but also faces significant practical hurdles. Because the RNAi effect is cell autonomous (i.e., siRNAs will only knock down genes in cells in which they are expressed), at least in mammalian cells, successful therapy of cancer will require expression of siRNAs in all malignant cells of a tumor. This will likely prove to be very challenging, if not impossible, in vivo, and RNAi will thus likely have an adjuvant rather than a primary role in cancer therapy. This is unfortunate because the exquisite specificity of RNAi might otherwise be exploited by selectively knocking down the expression of mutant oncogene mRNA while leaving normal allele RNA intact (Cancer Cell 2002;2:243–247). For treating dominant genetic diseases, such as alpha-1 antitrypsin deficiency, expression in all hepatocytes is not needed, and this approach may still be feasible.

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Successful clinical application of RNAi for gene therapy of chronic hepatitis, liver cancer, and dominant genetic diseases of the liver will require the development of more efficient and safe methods of gene delivery (Clin Liver Dis 2001;5:381– 414). Towards this goal, there has been an explosion of work focused on potential experimental and therapeutic applications of RNAi technology, and we will undoubtedly soon see publications using various vectors to deliver and express siRNA molecules in vivo (Nat Biotechnol 2002;20:1006 –1010, RNA 2003;9:493–501). An increasing number of biotechnology companies are also providing reagents for RNAi, and if the rapidity with which this technology has been commercialized speaks at all to its ultimate impact, RNAi will be very a important area of investigation. As further testimony to the far-reaching impact of this area of research, Science named small RNAs, including those involved in RNAi, “Breakthrough of the Year” for 2002 (Science 2002;298: 2296 –2297). Whether RNAi will ever play a direct role in the treatment of patients with hepatitis C is currently unclear given the steady progress in the development of small molecule inhibitors of various HCV proteins (Hepatology 2002;35:224 –231, Nat Rev Drug Discov 2002;1:867– 881). Nonetheless, the discovery of RNAi and development of siRNA vectors promise to revolutionize functional genomics and will undoubtedly contribute enormously to our understanding of liver biology in health and disease. TIMOTHY J. DAVERN, M.D.