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With RNA Interference, Silence Is Golden

With RNA Interference, Silence Is Golden Clinical researchers are constantly trying to convince cells within the body to change their ways. In some conditions, such as cancer, they want to stop cells from growing; in others, such as Parkinson disease, they want to keep cells from dying. Because gene expression controls how a cell looks and acts, a logical approach to manipulating a cell's behavior would be to turn genes on or off. But effectively targeting a particular gene within a chromosome is a laborious process, and it is not even currently feasible for some genes. So researchers are studying a variety of other strategies they hope will achieve the same ends. The gene silencing effect of RNA interference is demonstrated with images of light (red, most intense luciferase expression; blue, least intense) emitted from mice co-transfected with luciferase DNA and either no siRNA, at left, or luciferase siRNA, at right (Nature. 2002; 418:38-39). (Photo credit: Nature Publishing Group) Altering cell behavior Altering cell behavior In cancer research, scientists have had varying degrees of success with approaches aimed at altering the cancer cells' environment. For example, some are attacking the blood vessels that nourish a tumor with drugs that block angiogenesis (JAMA. 2003;290:2529-2533). Others are looking at ways to manipulate surrounding normal tissues to keep cancer cells from metastasizing (JAMA. 2004;291:2419-2421). Altering cell behavior Researchers in many fields are also looking into strategies to target cellular proteins, such as transcription factors and cell surface receptors. For instance, the transcription factor nuclear factor-κB is an attractive target because it plays a role in inflammation and other processes. And scientists have found that blocking major histocompatibility molecules on cell surfaces can have an effect on the immune response. Altering cell behavior But a new concept aimed at regulating the behavior of cells is now gaining momentum: RNA interference (RNAi). RNAi is a form of gene silencing that in no way involves the gene itself. Instead, the messenger RNA transcribed from the gene is attacked and destroyed, thereby obliterating the gene's message before it is translated into the protein. Scientists are finding the technique to be useful in a wide variety of applications, many of which may someday have clinical utility. Knocking down, not out Knocking down, not out The concept of RNAi is simple: deliver a double-stranded small interfering RNA (siRNA) into a cell, and it will bind to its complementary messenger RNA. It then calls over a cluster of proteins that are part of an RNA-induced silencing complex that degrades the messenger RNA. RNAi occurs naturally in humans and other mammals, as well as in other organisms such as plants and worms. It likely plays a diverse role throughout life—from controlling gene expression during development and aging to protecting cells against invading viruses (Trends Biochem Sci. 2003;28:196-201). Knocking down, not out Although the effect of gene silencing with RNAi is similar to that of knocking out a gene at the DNA level—a strategy used to learn about the function of a specific gene by silencing its expression—there are several differences. RNAi gives researchers more flexibility than gene knockouts because it can allow for fine-tuning of gene expression. This may be important for modulating so-called essential genes, which cannot be knocked out in animal models without killing the animal. And in many instances, cells in a disease state overexpress a normal gene, so decreasing expression considerably but not completely would be desirable. Knocking down, not out "In many situations, two-fold, three-fold, or five-fold changes are more than enough in a gene product to show a physiological effect," said Phillip Sharp, PhD, of the Massachusetts Institute of Technology, in Cambridge, who shared the 1993 Nobel Prize in Physiology or Medicine. Knocking down, not out RNAi is also reversible. If a nonreversible effect is the goal, however, genes that encode double-stranded RNA can be inserted into cells so that heritable gene silencing is achieved, making the RNAi essentially a form of gene therapy. Further work is needed, however, to perfect the technique's ability to fine-tune gene expression. A window into gene function A window into gene function One of RNAi's most attractive qualities is that it can help answer a number of scientific questions relating to gene discovery and function. A window into gene function "Right now, the clear success with RNAi is that it's being used by all kinds of research laboratories to manipulate expression of particular genes to understand function," said Henry Paulson, PhD, of the University of Iowa, in Iowa City. A window into gene function Greg Hannon, PhD, of the Cold Spring Harbor Laboratory, in Cold Spring Harbor, NY, recently created the first library of human RNAi clones, a collection of siRNAs corresponding to every gene in the genome (Nature. 2004;428:427-431). His own laboratory group is using the library to identify potential new targets for cancer therapeutics. A window into gene function Other researchers are using siRNAs as potential gene silencing therapies, although many of these studies are preliminary, said Hannon. "There are still substantial problems to overcome before we see RNAi as a therapy," he said. "Of course, if they are overcome, then this will be really quite revolutionary." Clinical hurdles Clinical hurdles Delivering siRNA into cells is the biggest impediment to RNAi therapy. "It's clear that if you have siRNA in a cell, it will effectively silence a gene and potentially have a therapeutic impact," said Sharp. "So then you're left with [the question] can you get it there? And that's the question that a lot of people are working on in a variety of different settings." Clinical hurdles So far, no single delivery strategy looks particularly promising, although Sharp is optimistic that scientists will find a way to overcome this hurdle. "It is possible that there will be a new technology such as a chemical modification, a new carrier material, or a new delivery method that will solve the problem," he said. Clinical hurdles Sharp suspects, however, that finding a single optimal delivery system for all applications is unlikely. "Delivery to the lung is different from delivery to the liver or to the joints or to the eye, so delivery systems may emerge to be tissue-specific," he said. Clinical hurdles Optimizing the binding properties of siRNAs within cells and minimizing any adverse effects also need to be addressed through in vivo studies. It is also time to move beyond using RNAi in cell culture studies, said Paulson. Clinical hurdles "We and many other groups are in a position now where we can start to [study RNAi] in a mouse model and see whether it is tolerable and effective and sustained in its effect," he said. Some in vivo experiments have already shown promising results, particularly in HIV and cancer research (Nat Rev Mol Cell Biol. 2003;4:457-467). Clinical potential Clinical potential Despite the long road ahead in preclinical studies of the technique, many scientists believe that it is just a matter of time before RNAi enters the clinical realm. "Assuming that delivery problems can be settled, I think the clinical indications are pretty wide open," said Judy Lieberman, MD, PhD, of Harvard Medical School, in Boston. Clinical potential Lieberman has shown that combined RNAi targeting of CCR5, the major HIV-1 coreceptor in macrophages, and p24, an HIV structural protein, is able to abolish HIV infection in macrophages (J Virol. 2003;77:7174-7181). She also demonstrated that using RNAi to silence the apoptosis-promoting Fas gene inhibited hepatocyte death in a mouse model of hepatitis. Because the Fas gene is implicated in a variety of liver diseases (including viral, autoimmune, and transplant rejection hepatitis), the approach holds promise in preventing liver injury (Nat Med. 2003;9:347-351). Clinical potential Paulson is collaborating with Beverly Davidson, PhD, in studies using RNAi to target neurodegenerative conditions such as Alzheimer disease and dystonia. "The idea is that in many of these diseases, there's a dominant acting toxic gene," said Paulson. "If we were able to eliminate expression of that toxic copy and keep the normal copy going, that would be great." Clinical potential Pharmaceutical companies are also showing interest in the technology—researchers from more than a dozen delivered scientific presentations at a conference in Boston last month. According to Sharp, most, if not all, are using RNAi in their search for drug targets. Many are also interested in its potential as a therapy itself. Clinical potential "I don't think there is a condition that the pharmaceutical world would consider ‘untargetable' if you could make this technology work," Sharp said. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png JAMA American Medical Association

With RNA Interference, Silence Is Golden

Hampton, Tracy
JAMA , Volume 291 (23) – Jun 16, 2004
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Publisher
American Medical Association
Copyright
Copyright © 2004 American Medical Association. All Rights Reserved.
ISSN
0098-7484
eISSN
1538-3598
DOI
10.1001/jama.291.23.2803
Publisher site
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Abstract

Clinical researchers are constantly trying to convince cells within the body to change their ways. In some conditions, such as cancer, they want to stop cells from growing; in others, such as Parkinson disease, they want to keep cells from dying. Because gene expression controls how a cell looks and acts, a logical approach to manipulating a cell's behavior would be to turn genes on or off. But effectively targeting a particular gene within a chromosome is a laborious process, and it is not even currently feasible for some genes. So researchers are studying a variety of other strategies they hope will achieve the same ends. The gene silencing effect of RNA interference is demonstrated with images of light (red, most intense luciferase expression; blue, least intense) emitted from mice co-transfected with luciferase DNA and either no siRNA, at left, or luciferase siRNA, at right (Nature. 2002; 418:38-39). (Photo credit: Nature Publishing Group) Altering cell behavior Altering cell behavior In cancer research, scientists have had varying degrees of success with approaches aimed at altering the cancer cells' environment. For example, some are attacking the blood vessels that nourish a tumor with drugs that block angiogenesis (JAMA. 2003;290:2529-2533). Others are looking at ways to manipulate surrounding normal tissues to keep cancer cells from metastasizing (JAMA. 2004;291:2419-2421). Altering cell behavior Researchers in many fields are also looking into strategies to target cellular proteins, such as transcription factors and cell surface receptors. For instance, the transcription factor nuclear factor-κB is an attractive target because it plays a role in inflammation and other processes. And scientists have found that blocking major histocompatibility molecules on cell surfaces can have an effect on the immune response. Altering cell behavior But a new concept aimed at regulating the behavior of cells is now gaining momentum: RNA interference (RNAi). RNAi is a form of gene silencing that in no way involves the gene itself. Instead, the messenger RNA transcribed from the gene is attacked and destroyed, thereby obliterating the gene's message before it is translated into the protein. Scientists are finding the technique to be useful in a wide variety of applications, many of which may someday have clinical utility. Knocking down, not out Knocking down, not out The concept of RNAi is simple: deliver a double-stranded small interfering RNA (siRNA) into a cell, and it will bind to its complementary messenger RNA. It then calls over a cluster of proteins that are part of an RNA-induced silencing complex that degrades the messenger RNA. RNAi occurs naturally in humans and other mammals, as well as in other organisms such as plants and worms. It likely plays a diverse role throughout life—from controlling gene expression during development and aging to protecting cells against invading viruses (Trends Biochem Sci. 2003;28:196-201). Knocking down, not out Although the effect of gene silencing with RNAi is similar to that of knocking out a gene at the DNA level—a strategy used to learn about the function of a specific gene by silencing its expression—there are several differences. RNAi gives researchers more flexibility than gene knockouts because it can allow for fine-tuning of gene expression. This may be important for modulating so-called essential genes, which cannot be knocked out in animal models without killing the animal. And in many instances, cells in a disease state overexpress a normal gene, so decreasing expression considerably but not completely would be desirable. Knocking down, not out "In many situations, two-fold, three-fold, or five-fold changes are more than enough in a gene product to show a physiological effect," said Phillip Sharp, PhD, of the Massachusetts Institute of Technology, in Cambridge, who shared the 1993 Nobel Prize in Physiology or Medicine. Knocking down, not out RNAi is also reversible. If a nonreversible effect is the goal, however, genes that encode double-stranded RNA can be inserted into cells so that heritable gene silencing is achieved, making the RNAi essentially a form of gene therapy. Further work is needed, however, to perfect the technique's ability to fine-tune gene expression. A window into gene function A window into gene function One of RNAi's most attractive qualities is that it can help answer a number of scientific questions relating to gene discovery and function. A window into gene function "Right now, the clear success with RNAi is that it's being used by all kinds of research laboratories to manipulate expression of particular genes to understand function," said Henry Paulson, PhD, of the University of Iowa, in Iowa City. A window into gene function Greg Hannon, PhD, of the Cold Spring Harbor Laboratory, in Cold Spring Harbor, NY, recently created the first library of human RNAi clones, a collection of siRNAs corresponding to every gene in the genome (Nature. 2004;428:427-431). His own laboratory group is using the library to identify potential new targets for cancer therapeutics. A window into gene function Other researchers are using siRNAs as potential gene silencing therapies, although many of these studies are preliminary, said Hannon. "There are still substantial problems to overcome before we see RNAi as a therapy," he said. "Of course, if they are overcome, then this will be really quite revolutionary." Clinical hurdles Clinical hurdles Delivering siRNA into cells is the biggest impediment to RNAi therapy. "It's clear that if you have siRNA in a cell, it will effectively silence a gene and potentially have a therapeutic impact," said Sharp. "So then you're left with [the question] can you get it there? And that's the question that a lot of people are working on in a variety of different settings." Clinical hurdles So far, no single delivery strategy looks particularly promising, although Sharp is optimistic that scientists will find a way to overcome this hurdle. "It is possible that there will be a new technology such as a chemical modification, a new carrier material, or a new delivery method that will solve the problem," he said. Clinical hurdles Sharp suspects, however, that finding a single optimal delivery system for all applications is unlikely. "Delivery to the lung is different from delivery to the liver or to the joints or to the eye, so delivery systems may emerge to be tissue-specific," he said. Clinical hurdles Optimizing the binding properties of siRNAs within cells and minimizing any adverse effects also need to be addressed through in vivo studies. It is also time to move beyond using RNAi in cell culture studies, said Paulson. Clinical hurdles "We and many other groups are in a position now where we can start to [study RNAi] in a mouse model and see whether it is tolerable and effective and sustained in its effect," he said. Some in vivo experiments have already shown promising results, particularly in HIV and cancer research (Nat Rev Mol Cell Biol. 2003;4:457-467). Clinical potential Clinical potential Despite the long road ahead in preclinical studies of the technique, many scientists believe that it is just a matter of time before RNAi enters the clinical realm. "Assuming that delivery problems can be settled, I think the clinical indications are pretty wide open," said Judy Lieberman, MD, PhD, of Harvard Medical School, in Boston. Clinical potential Lieberman has shown that combined RNAi targeting of CCR5, the major HIV-1 coreceptor in macrophages, and p24, an HIV structural protein, is able to abolish HIV infection in macrophages (J Virol. 2003;77:7174-7181). She also demonstrated that using RNAi to silence the apoptosis-promoting Fas gene inhibited hepatocyte death in a mouse model of hepatitis. Because the Fas gene is implicated in a variety of liver diseases (including viral, autoimmune, and transplant rejection hepatitis), the approach holds promise in preventing liver injury (Nat Med. 2003;9:347-351). Clinical potential Paulson is collaborating with Beverly Davidson, PhD, in studies using RNAi to target neurodegenerative conditions such as Alzheimer disease and dystonia. "The idea is that in many of these diseases, there's a dominant acting toxic gene," said Paulson. "If we were able to eliminate expression of that toxic copy and keep the normal copy going, that would be great." Clinical potential Pharmaceutical companies are also showing interest in the technology—researchers from more than a dozen delivered scientific presentations at a conference in Boston last month. According to Sharp, most, if not all, are using RNAi in their search for drug targets. Many are also interested in its potential as a therapy itself. Clinical potential "I don't think there is a condition that the pharmaceutical world would consider ‘untargetable' if you could make this technology work," Sharp said.

Journal

JAMA – American Medical Association

Published: Jun 16, 2004

Keywords: rna interference,genes

There are no references for this article.