Is siRNA the tool of the future for in vivo mammalian gene research? The experts speak out.

RNA INTERFERENCE using short, double-stranded RNA (small interfering RNA, or siRNA) was recently introduced for suppressing gene expression in mammalian cells. Although the molecular mechanism of siRNA is not completely understood, it is generally believed that siRNA induces the degradation of target mRNA in a sequence-specific manner, leading to posttranscriptional silencing of gene expression. As such, siRNA could potentially be used both as a research tool and as a therapeutic approach. Its efficiency also recommends it, since siRNA has been shown by several studies to be more efficient than antisense oligonucleotides, another method commonly used to reduce protein expression (8, 10, 19). The availability of genome-scale collections of siRNAs further increases the attractiveness of this approach (3, 20, 23). Numerous studies published in the last couple of years have unequivocally demonstrated the effectiveness of siRNA in various types of cultured mammalian cells (5, 6, 7, 14, 16, 24). Important potential uses for the technology include characterization of known genes by knocking out a given gene and determining whether the pathway responds in a way that is consistent with a given hypothesis, identification of novel genes and target identification and validation by sequentially knocking out genes and observing the responses, and for use in drug discovery and drug screening. Potential therapeutic applications are also being considered. Yet even as siRNA enters the mainstream of biomedicine research, questions remain as to what the range of its capabilities may be for eliciting information about how specific genes function in mammals in vivo. The potential utility of siRNA for studying mammals in vivo is of particular importance, since it will likely determine the long-term viability of the siRNA approach. We asked several researchers in the field where they see the present and the future of RNA interference and siRNA as tools for research into the physiology of intact mammals and what their limitations might be. Their responses follow. Does siRNA work in mammals in vivo? Gordon Mitchell, who is chairman of the Department of Comparative Biosciences, School of Veterinary Medicine at the University of Wisconsin, gives the answer as a definitive yes. “We have done it and published it. It is in the rat spinal cord in vivo” (1, 2). His laboratory is devoted to studies of plasticity in respiratory control, with a major emphasis on neuroplasticity elicited by intermittent hypoxia and spinal plasticity following spinal cord injury. Mitchell said, “Our laboratory was one of the first to apply the use of siRNA to the mammalian nervous system in vivo. In this case, we did not use the siRNA to disrupt transcription or to degrade the target mRNA. Instead, we took advantage of the property of siRNAs to inhibit translation of existing mRNA. We demonstrated the efficacy of this approach within hours (at a time when we do not expect significant degradation of the mRNA) by demonstrating that new protein synthesis was impaired. In this case, the protein in question was brain-derived trophic factor, or BDNF. We took advantage of the lack of RNases in the cerebrospinal fluid. We used a lipophilic transfection reagent (Oligofectamine) and delivered the siRNA duplexes directly to the cerebrospinal fluid of the spinal cord of rats.” The utility of siRNA in interrupting transcriptions of specific genes in living systems is excellent, agreed Asrar B. Malik, Distinguished Professor and Head of the Department of Pharmacology at the College of Medicine, University of IllinoisChicago. His lab, which studies vascular and lung biology, uses liposomes to deliver siRNA to the appropriate site (21). Patty J. Lee, who is Assistant Professor of Internal Medicine-Pulmonary at Yale University School of Medicine, is using siRNA to study the mechanism of lung injury. Her lab is using carrier agents such as Lipofectamine for delivery in cells. For animals, “we have used regular/unaltered siRNA and will soon be using a viral delivery system as well as direct injection/ nasal administration of ‘stabilized’ constructs,” she said (25). Both these methods have proved effective for them. According to Lee, “To the best of our knowledge, we are the first to demonstrate that lung-specific siRNA delivery can be achieved by intranasal administration without the need for viral vectors or transfection agents in vivo, thereby obviating potential concerns for toxicity if siRNA technology is to have clinical application in the future.” Joseph Verbalis is Professor of Medicine and Physiology and Director of the General Research Center at Georgetown University Medical Center. His lab is investigating the role that alterations in vasopressin V2 receptor expression play in regulation of vasopressin-mediated antidiuresis. They have tried to use siRNA to knock down this receptor subtype using intravenously injected chemically synthesized siRNA with the assistance of the transfection reagent 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). “While we have not had as much experience with siRNA,” he said, “we feel it will be more effective and more reliable in downregulating the expression of proteins.” Antisense was ineffective for the work they are doing in the brain, he said, because the results tended to be highly variable and not very predictable in decreasing the expression of given receptor proteins. According to Ali Hassan, who works with Verbalis, “This siRNA/DOTAP approach has been reasonably successful for us; we saw a 50% reduction in V2 receptor expression following transfection, and additionally this knockdown had a physiological effect. Following siRNA infusion treatment with a V2 receptor-specific antagonist (dDAVP), we saw significantly increased urine volume and decreased osmolality, consistent with V2 receptor knockdown” (11, 12). Tina Chabrashvili’s lab at Georgetown is also using synthetic siRNA duplex in vivo in the rat. “We have been applying hydrodynamics-based transfection by systemic administration in vivo, which is basically a ‘high-pressure delivery’ technique to deliver siRNA duplex,” she said. They have also tried to deliver siRNA using plasmids and expression cassettes with Physiol Genomics 18: 252–254, 2004; 10.1152/physiolgenomics.00146.2004.