RNA Interference as Potential Therapy — Not So Fast
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《新英格兰医药杂志》
The 1989 Nobel Prize in Chemistry was awarded to Sidney Altman and Thomas Cech for the discovery that RNA is not only a molecule of heredity, it is also a biocatalyst. The Royal Swedish Academy of Sciences offered a prescient comment when it announced the award1: the "future use of gene shears will require that we learn more about the molecular mechanisms . . . of RNA." The discovery of RNA interference (RNAi),2 an evolutionarily conserved mechanism that silences gene expression, underscores this point. Perhaps even more exciting than its discovery is the specter of harnessing this robust and specific gene-silencing mechanism as a therapeutic tool. Several animal models of human disease have been successfully treated with the use of RNAi. A recent report by Grimm et al.3 affirms the promise of the method in the in vivo setting, but it also serves as a sobering reminder that the design of safe and effective RNA-based gene therapies will depend on a comprehensive understanding of the endogenous molecular mechanisms involved.
RNAi is mediated by short double-stranded RNA. The sequence-specific degradation of messenger RNA (mRNA) is elicited by the base pairing of complementary RNA strands, each approximately 22 nucleotides in length (Figure 1). These molecular complexes, which are termed small interfering RNA (siRNA) duplexes, are generated in the cytoplasm, either through the cleavage of endogenous long double-stranded RNA or from synthetic short hairpin RNA (shRNA). The production of this synthetic shRNA from gene-therapy vectors (either viral or nonviral) is an efficient means of experimentally eliciting RNAi in vivo.
Figure 1. Silencing RNA: More May Not Be Better.
Pre-microRNA (pre-miRNA) is an endogenous product of the transcription of host genomic DNA. Short hairpin RNA (shRNA) is a product of the transcription of exogenous viral-vector DNA. Both types of transcription occur in the nucleus. Both pre-miRNA and shRNA are destined for the cytoplasm, where they are processed by an enzyme called Dicer. Exportin-5 is a key component of the machinery used to export both molecules from the nucleus; it can limit the rate of export. A recent study by Grimm et al.3 showed that the overexpression of potential therapeutic shRNA can saturate the export machinery, leading to competition with the normal export of endogenous miRNA precursors — which may explain the lethality of some shRNA. In the cytoplasm, microRNA (miRNA) is involved in the inhibition of the translation of target mRNA. The guide strand of small interfering RNA (siRNA), derived from shRNA precursors, is incorporated into the RNA-induced silencing complex (RISC) and is involved in the degradation of specific target mRNA.
How does RNAi change gene expression? One of the RNA strands in the siRNA duplex, termed the guide strand, becomes incorporated into the RNA-induced silencing complex (RISC). The second strand, the passenger strand, is ejected from the RISC complex. The guide strand provides exquisite specificity to RISC, which binds and then degrades complementary target mRNA in the cytoplasm (and possibly in the nucleus) (Figure 1).
A related endogenous pathway, involving microRNA (miRNA), also exists in mammalian cells.4 Hundreds of discrete regions within the genome encode atypical genes that give rise to miRNA. Although they are transcribed by RNA polymerase II, the enzyme that typically produces mRNA destined for translation into proteins, these atypical genes do not encode a protein. Instead, they produce RNA species that regulate the translation of other proteins. Most endogenous miRNA functions as a sophisticated conductor of genetic pathways by manipulating the translational regulation of many genes at the same time. The specificity of this manipulation is relatively low, because target RNA species contain sequences that are only roughly related to the complementary miRNA sequence. Bioinformatics analyses suggest that up to 30 percent of human genes may be regulated by miRNA.5 In brief, RNAi and miRNA both result in decreased levels of functional protein within cells, but RNAi tends to affect steady-state mRNA levels, whereas miRNA usually affects the efficiency with which mRNA is translated into protein.4
Grimm et al. evaluated the efficacy of long-term RNAi-based therapeutic agents in the livers of adult mice. They used 49 distinct constructs consisting of viral DNA that encode synthetic shRNA species directed against six unique target mRNA species. Thirty-six constructs resulted in concentration-dependent liver toxicity, with premature death occurring in 23 of the mouse models. The toxic effects were not restricted to the specific shRNA expressed or to the mRNA target against which it was designed, nor were they related to components of the vector or a consequence of the activation of innate defense pathways against double-stranded RNA (which seem to have evolved as antiviral responses). Whether these findings are relevant to other types of tissue and organs will need to be addressed in future studies.
Precursor RNA species from which miRNA is derived, termed pre-miRNA, require processing to produce biologically active miRNA. Pre-miRNA is similar in structure to exogenous shRNA derived from viral gene-therapy vectors. Especially relevant, Grimm et al. argue, is that the export of shRNA and pre-miRNA from the nucleus has a common mediator, called exportin-5. It seems that cells transfected with vectors containing shRNA produced sufficient synthetic shRNA in the nucleus to block the normal export of endogenous pre-miRNA from the nucleus. In other words, the two structurally related molecules had to compete for a rate-limiting nuclear-export pathway. Consistent with this explanation is the finding by Grimm et al. that increases in the cellular levels of exportin-5 reduced the in vivo hepatotoxicity observed in mice treated with shRNA-containing vectors.
Approaches involving RNAi have revolutionized the interruption of gene function and have been used as an experimental tool in vitro, and they offer hope for in vivo therapy in humans. Will the study by Grimm et al. enhance the development of RNAi-based therapeutic agents? Only time will tell, but Altman and Cech would argue that a better understanding of endogenous cellular RNA pathways cannot hurt.
No potential conflict of interest relevant to this article was reported.
Source Information
From the Division of Nephrology, St. Michael's Hospital and the University of Toronto, Toronto.
References
Ribonucleic acid (RNA) — a biomolecule of many functions. Press release of the Royal Swedish Academy of Sciences, Stockholm, October 12, 1989.
Fire 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;391:806-811.
Grimm D, Streetz KL, Jopling CL, et al. Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 2006;441:537-541.
Valencia-Sanchez MA, Liu J, Hannon GJ, Parker R. Control of translation and mRNA degradation by miRNAs and siRNAs. Genes Dev 2006;20:515-524.
Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 2005;120:15-20.(Philip A. Marsden, M.D.)
RNAi is mediated by short double-stranded RNA. The sequence-specific degradation of messenger RNA (mRNA) is elicited by the base pairing of complementary RNA strands, each approximately 22 nucleotides in length (Figure 1). These molecular complexes, which are termed small interfering RNA (siRNA) duplexes, are generated in the cytoplasm, either through the cleavage of endogenous long double-stranded RNA or from synthetic short hairpin RNA (shRNA). The production of this synthetic shRNA from gene-therapy vectors (either viral or nonviral) is an efficient means of experimentally eliciting RNAi in vivo.
Figure 1. Silencing RNA: More May Not Be Better.
Pre-microRNA (pre-miRNA) is an endogenous product of the transcription of host genomic DNA. Short hairpin RNA (shRNA) is a product of the transcription of exogenous viral-vector DNA. Both types of transcription occur in the nucleus. Both pre-miRNA and shRNA are destined for the cytoplasm, where they are processed by an enzyme called Dicer. Exportin-5 is a key component of the machinery used to export both molecules from the nucleus; it can limit the rate of export. A recent study by Grimm et al.3 showed that the overexpression of potential therapeutic shRNA can saturate the export machinery, leading to competition with the normal export of endogenous miRNA precursors — which may explain the lethality of some shRNA. In the cytoplasm, microRNA (miRNA) is involved in the inhibition of the translation of target mRNA. The guide strand of small interfering RNA (siRNA), derived from shRNA precursors, is incorporated into the RNA-induced silencing complex (RISC) and is involved in the degradation of specific target mRNA.
How does RNAi change gene expression? One of the RNA strands in the siRNA duplex, termed the guide strand, becomes incorporated into the RNA-induced silencing complex (RISC). The second strand, the passenger strand, is ejected from the RISC complex. The guide strand provides exquisite specificity to RISC, which binds and then degrades complementary target mRNA in the cytoplasm (and possibly in the nucleus) (Figure 1).
A related endogenous pathway, involving microRNA (miRNA), also exists in mammalian cells.4 Hundreds of discrete regions within the genome encode atypical genes that give rise to miRNA. Although they are transcribed by RNA polymerase II, the enzyme that typically produces mRNA destined for translation into proteins, these atypical genes do not encode a protein. Instead, they produce RNA species that regulate the translation of other proteins. Most endogenous miRNA functions as a sophisticated conductor of genetic pathways by manipulating the translational regulation of many genes at the same time. The specificity of this manipulation is relatively low, because target RNA species contain sequences that are only roughly related to the complementary miRNA sequence. Bioinformatics analyses suggest that up to 30 percent of human genes may be regulated by miRNA.5 In brief, RNAi and miRNA both result in decreased levels of functional protein within cells, but RNAi tends to affect steady-state mRNA levels, whereas miRNA usually affects the efficiency with which mRNA is translated into protein.4
Grimm et al. evaluated the efficacy of long-term RNAi-based therapeutic agents in the livers of adult mice. They used 49 distinct constructs consisting of viral DNA that encode synthetic shRNA species directed against six unique target mRNA species. Thirty-six constructs resulted in concentration-dependent liver toxicity, with premature death occurring in 23 of the mouse models. The toxic effects were not restricted to the specific shRNA expressed or to the mRNA target against which it was designed, nor were they related to components of the vector or a consequence of the activation of innate defense pathways against double-stranded RNA (which seem to have evolved as antiviral responses). Whether these findings are relevant to other types of tissue and organs will need to be addressed in future studies.
Precursor RNA species from which miRNA is derived, termed pre-miRNA, require processing to produce biologically active miRNA. Pre-miRNA is similar in structure to exogenous shRNA derived from viral gene-therapy vectors. Especially relevant, Grimm et al. argue, is that the export of shRNA and pre-miRNA from the nucleus has a common mediator, called exportin-5. It seems that cells transfected with vectors containing shRNA produced sufficient synthetic shRNA in the nucleus to block the normal export of endogenous pre-miRNA from the nucleus. In other words, the two structurally related molecules had to compete for a rate-limiting nuclear-export pathway. Consistent with this explanation is the finding by Grimm et al. that increases in the cellular levels of exportin-5 reduced the in vivo hepatotoxicity observed in mice treated with shRNA-containing vectors.
Approaches involving RNAi have revolutionized the interruption of gene function and have been used as an experimental tool in vitro, and they offer hope for in vivo therapy in humans. Will the study by Grimm et al. enhance the development of RNAi-based therapeutic agents? Only time will tell, but Altman and Cech would argue that a better understanding of endogenous cellular RNA pathways cannot hurt.
No potential conflict of interest relevant to this article was reported.
Source Information
From the Division of Nephrology, St. Michael's Hospital and the University of Toronto, Toronto.
References
Ribonucleic acid (RNA) — a biomolecule of many functions. Press release of the Royal Swedish Academy of Sciences, Stockholm, October 12, 1989.
Fire 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;391:806-811.
Grimm D, Streetz KL, Jopling CL, et al. Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 2006;441:537-541.
Valencia-Sanchez MA, Liu J, Hannon GJ, Parker R. Control of translation and mRNA degradation by miRNAs and siRNAs. Genes Dev 2006;20:515-524.
Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 2005;120:15-20.(Philip A. Marsden, M.D.)