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

Beverly Ventura

Managing Editor, Physiological Genomics


    INTRODUCTION
 TOP
 INTRODUCTION
 REFERENCES
 
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 Illinois-Chicago. 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 the tissue-specific promoter in vivo but don’t have conclusive results yet, she said. "It’s a really crucial discovery that has provided a reliable, robust reverse genetics tool for the future."

Peter Sandy is a postdoctoral fellow in the laboratory of Luk Van Parijs at the Center for Cancer Research at the Massachusetts Institute of Technology. The main focus of their lab is on understanding the molecular mechanisms that govern immune function during physiological and pathological conditions. His lab at MIT pioneered lentivirus-based siRNA (22). "My colleagues developed a lentiviral vector (pLL3.7) that expresses shRNAs (short hairpin RNA) simultaneously with the GFP (green fluorescent protein) marker, which can be used to track transfected/infected cells." The advantage of using lentiviruses, he said, is that they can infect a broad range of cell types (including nondividing, postmitotic, and primary cells) and that integrating into the host genome can allow for stable expression of the transgenes.

What are the strengths and weaknesses of using siRNA in mammals in vivo?
According to Sandy, by using lentiviruses as delivery tools for shRNAs, it is possible to generate knockdown animals in a few weeks, rather than a few years, which is what is normally required for homologous recombination-based gene targeting (knockout). Sandy also considers the relative simplicity and inexpensiveness as strengths of the siRNA approach. He pointed out that "RNAi is probably the right choice for the generation of ‘hypomorphic alleles’ that may efficiently model gene dosage effects, typical of several human pathologies."

Hassan said that the differences between siRNA and conventional gene knockouts are that siRNA "allows for the knockdown of a specific protein in adult animals which have developed normally. In knockout mice, developmental effects are often profound, and while this can in itself provide interesting information, it can make the animals poor models for physiological studies. Secondly, RNAi could, theoretically, be easily applied to any vertebrate and is not practically limited to mice."

Use of this technology is "rapidly improving, with increased stability and efficiency of the constructs," said Lee. In the future, "tissue targeting should be attempted."

Many investigators in the field, however, acknowledge the challenges in using siRNA in mammals in vivo. "For in vivo application, siRNA faces many of the same challenges seen in the somatic gene delivery field, the most prominent of which is how to get siRNA into the target cells," said Mingyu Liang, Assistant Professor of Physiology at the Medical College of Wisconsin (15).

"Delivery methods should be improved, and there is a great demand (and promising results) for conditional and tissue-specific shRNA expression," Sandy agreed. Lee added that the minimal effective doses are still not optimal. "Using tissue-specific promoters or using inducible promoters—that’s going to be the way to do that," Chabrashvili said. She also noted that siRNA molecules that don’t bind as well to RISC ("RNA-induced silencing compound") are more readily released and fairly rapidly degraded, and she suggested that stability could be improved by modification of carbohydrate into nucleotide bond and base modifications.

Liang said that the technical similarity between in vivo siRNA and somatic gene delivery "implies that a lot of experience gained from gene delivery studies might be helpful for in vivo siRNA studies." Hassan is also optimistic, "Many of the problems of in vivo application have already been solved, and the remaining ones appear solvable."

Sandy noted that "off-targeting" of siRNA can have undesired effects, which is "particularly relevant for considering RNAi as a therapeutic tool." Indeed, several studies have shown that partial matches are sufficient for inducing gene knockdown. Some siRNAs have even been shown to induce an interferon response, a concern that was once associated only with longer double-stranded RNA. As the first line of defense against false conclusions, Liang emphasized that "appropriate controls are critical for a siRNA experiment just as they are in any other experiments." Usage of negative controls is mandatory, agreed Chabrashvili.

Where do these researchers see the technology going in the future?
"It is new territory, particularly when applied to vertebrates in vivo," said Mitchell. "It is far from certain what therapeutic applications will arise from the use of siRNAs in vivo, but it seems highly likely that interesting applications will be found." Although they have only preliminary data to date, he said his lab is developing the technique of delivering siRNAs targeted against BDNF directly to targeted neurons in vivo. They are also developing other molecular targets for their in vivo studies of neuroplasticity in the mammalian spinal cord. Malik of University of Illinois-Chicago agreed that it is hard to say where the technology will lead in the future without good clinical data, but he also feels the potential is high for it to make a positive impact.

Sandy believes that siRNA "will greatly facilitate the generation of particular disease models in mice and other model organisms (for example, rats or larger mammals), where knockout is difficult, time-consuming, or practically impossible" (13).

"I think it’s fair to say that application of RNAi to in vivo systems is in its infancy, to mammalian systems at least," Hassan said. "The technique has an enormous amount of potential, and there appear to be few barriers to its becoming a standard part of the physiologist’s toolbox." Hassan is particularly interested in incorporating viral methods of siRNA delivery and inducible expression of siRNA into his studies. He also believes that siRNA "provides an exceptionally useful tool to investigate the physiological role of alternative splicing."

"What we’ve done so far is a proof-of-concept," Verbalis added. "Antisense was great when it worked, but lots of times it didn’t work. Many people have abandoned antisense as a tool. It may be siRNA in vivo will be the same. But we’ve had exceptional in vitro success, and I personally believe we will be able to extrapolate it to use in vivo."

Michael McManus, who is a postdoctoral fellow in the lab of 1993 Nobel Prize winner Phillip Sharp of the Center for Cancer Research at MIT, also considers siRNA "extremely powerful, and can be used as a tool to create transient gene knockouts at the posttranscriptional level" (18). He pointed out that, "While biologists are excited about this new approach for suppressing gene expression, there is fascinating biology behind mammalian RNAi." In the MIT labs, they are using conditional knockout mice to unveil the role of mammalian Dicer, a factor that appears to be critical for naturally occurring RNAi that may involve endogenously generated micro RNAs, molecules that might reduce gene expression in much the same way as siRNAs (9, 17).

In vivo siRNA technology has aroused entrepreneurship as well, as evidenced by Fortune magazine naming siRNA biotech’s "next billion dollar breakthrough" in 2003. "We have filed a provisional patent for at least part of this work," said Mitchell, "and we are in the process of developing a new grant that relies extensively on this technology." Malik said they also have a patent pending, for intra-ocular delivery of siRNA. Indeed, clinical trials using siRNA to treat viral infection and other diseases are appearing on the horizon, the outcome of which will certainly have a tremendous impact on the fate of the entire siRNA field (4).

It is clear that siRNA-based gene silencing is still an evolving technology. It remains an open question whether the in vivo application of siRNA in mammals will revolutionize biomedical research and therapeutics or whether it is just one more tool with narrow utility. As McManus puts it, however, "it is almost certain that RNAi and related pathways will make a profound impact in biology in the coming decades."


    FOOTNOTES
 
10.1152/physiolgenomics.00146.2004.


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