From the Department of Pharmacology and Center for Basic Neuroscience, The University of Texas Southwestern Medical Center at Dallas.
A remarkable revolution is occurring in biomedical science that is likely to have profound implications for the way we treat human diseases in the future. In the past 2 years, we have learned that introduction of short double-stranded RNA (dsRNA) molecules into vertebrate cells can silence any gene homologous to the dsRNA. This process is called RNA interference (RNAi) and is based on the ability of the cell to recognize and degrade messenger RNAs (mRNAs) that base-pair with the antisense component of the dsRNA. RNA interference probably arose as a defense against viruses and transposable elements that replicate by means of dsRNA intermediates. This form of genetic immunity is ancient; organisms from paramecium to humans use similar mechanisms. In addition, it is now evident that RNAi normally regulates some cellular genes, and a whole host of noncoding antisense mRNAs are produced in cells that function to regulate other genes. Herein we discuss the link between the RNAi machinery and fragile X syndrome and the application of RNAi to functional genomic analysis, and we speculate about the future applications of this technology to the therapy for human neurological disease.
Biochemical and genetic experiments in Drosophila and Caenorhabitis elegans have provided most of our understanding of RNAi. Figure 1 shows a schematic illustration of our current understanding of this process. The RNAi is a posttranscriptional gene-silencing mechanism initiated by 21- to 23-nucleotide dsRNA fragments called small interfering RNAs (siRNAs). The siRNAs are produced by the endonuclease Dicer acting on long dsRNA molecules. Once produced, these siRNAs become associated with a nuclease complex called RNA-induced silencing complex (RISC) that contains a member of the conserved argonaute protein family, a helicase, and nucleases. The antisense component of the siRNA becomes associated with the RISC and is able to base-pair with homologous mRNAs in the cell, targeting them for cleavage and degradation by the RISC nucleases.1 The reduction in mRNA ultimately results in the specific loss of the protein encoded by that mRNA.
A model for RNA interference. A, The 21- to 23-nucleotide small interfering RNAs (siRNAs) are either chemically synthesized or produced endogenously by Dicer RNA nuclease III from long double-stranded RNA. These siRNAs are incorporated into a ribonucleoprotein complex called the RNA induced silencing complex (RISC) endonuclease. The RISC mediates sequence-specific target messenger RNA (mRNA) degradation by cleaving mRNAs that base-pair with the siRNA. AGO-2 (argonaute-2), FMR1 (fragile X mental retardation 1 gene product) protein (FMRP), and yet to be characterized proteins (?) are thought to be required for RISC formation. B, Endogenously delivered siRNA in the RNA interference pathway. Plasmid- or transgene-based expression (see Figure 2) of short hairpin RNA (shRNA) loops give rise to siRNAs in vivo after intracellular processing by Dicer.
In vertebrate cells, long dsRNA molecules activate a protein kinase that shuts off all protein synthesis in the cell, ultimately resulting in apoptotic cell death. However, RNAi mechanisms are still present in vertebrate cells and can be exploited by adding 21- to 23-nucleotide siRNA molecules directly to cells instead of long dsRNAs, thus bypassing the apoptotic response.2 This has opened vertebrate tissue culture cells to reverse genetics. The biochemical components mediating RNAi in invertebrates are also conserved in vertebrates, including homologues of Dicer, Rde-4, helicase, and argonaute depicted in Figure 1. However, many of the components required for RNAi have yet to be identified, and virtually nothing is understood about the regulation of this process.
Fragile X syndrome is a common form of inherited mental retardation caused by the loss of FMR1 (fragile X mental retardation 1 gene product) gene expression. In most cases, the disease is caused by the methylation-induced transcriptional silencing of the FMR1 gene that occurs as a result of expansion of CGG trinucelotide repeat in the 5′ untranslated region.3 Mouse knockout experiments indicate that loss of FMR1 protein (FMRP) is probably the underlying cause of fragile X syndrome phenotype. Intriguingly, mice lacking FMRP have abnormal dendritic spines on neurons.4 However, the pathogenic link between mental retardation and FMR1 is unclear.
The FMR1 gene encodes an RNA-binding protein (FMRP) that associates with ribosomes and acts to inhibit translation. Recently, a link between fragile X syndrome and RNAi was established. FMRP from cell lysates was identified as a component of a large protein complex that contains argonaute-2 (AGO-2), a protein normally present in the RISC complex required for RNA interference.5 In dsRNA-challenged Drosophila cells, FMRP co-immunoprecipitated a protein complex with RISC activity that contains siRNAs. These observations suggest that FMRP may be a component of RNAi machinery and raise the possibility that abnormal RNAi function in neurons lacking FMRP could underlie the phenotypes observed in patients with fragile X syndrome. However, additional studies will be required to firmly establish this link.
The sequencing of the human genome has demonstrated approximately 30 000 genes, hundreds of which may encode useful drug target candidates. The functional role most of these genes play is not known. How can we determine on which gene products to target therapeutic efforts? The use of RNAi is proving a simple, rapid way to begin to elucidate the role of these orphan genes in tissue culture cells. Adding siRNAs homologous to specific candidates can mimic loss-of-function mutations in those genes, providing vital clues about the normal roles of those gene products. This approach has now been used to mimic knockout mutations in every C elegans gene.6 For drug developers, RNAi phenotypes can provide clues about what to assay to screen antagonist drug candidates. These functional genomic efforts are likely to provide the medical community with new therapeutics to novel targets in the coming years.
For basic scientists interested in the roles specific genes play in intact animals, RNAi may provide an inexpensive alternative to knockout mice. Expressing transgenes encoding hairpin RNAs that can be processed by Dicer into siRNAs can allow specific genes to be targeted (Figure 1B). The best results have been observed with RNAi constructs driven by RNA polymerase III promoters like U6 and H1 (Figure 2).7,8 Most recently, introducing these RNAi transgenes into viral vectors may make it possible to target genes in intact animals.
Transgene-based RNA interference generating hairpin small interfering RNA. A polymerase (POL) III promoter (U6 or H1) drives the transcription of a 38- to 58-nucleotide RNA hairpin in cells carrying transgene. The transgene is composed of inverted repeats separated by a spacer of 3 to 9 nucleotides. Transcription of the transgene forms a double-stranded RNA that is processed by Dicer into small interfering RNA. The transcription is terminated by a polythymidine tract.
Can we use RNAi to directly treat human diseases? Investigators are actively pursuing the notion that RNAi can be adapted as an antiviral therapy, and transgenic or virally encoded RNAi constructs have great potential to selectively target disease genes that produce cancer and currently intractable neurological diseases. One of the unique features of RNAi is its exquisite sequence specificity. This high degree of specificity enables RNAi to selectively knock down expression of foreign genes and even mutant alleles of normal genes carrying point mutations, insertions, or deletions that commonly underlie human neurological diseases. Conceptually, RNAi technology can be used to curb diseases that are caused by dominantly acting mutant alleles, such as dominantly activating mutations in the ras gene that cause cancer, neurodegenerative diseases resulting from triplet expansion, and infectious diseases like AIDS. Table 1 shows recent work in which promising results have been obtained in applying RNAi to target genes producing human diseases. The siRNAs have been proven to successfully inhibit viral replication in cell culture.9 The siRNAs directed to the BCR/ABL fusion gene associated with the Philadelphia chromosome in chronic myeloid leukemia successfully killed leukemia cells but not control cells that do not express this fusion gene. Point mutation in the siRNA eliminated the effect.10 Recently, tissue culture models of the dominant genetic disorder spinobulbar muscular atrophy has been used to test the ability of RNAi to specifically down-regulate a human disease-related transcript. Spinobulbar muscular atrophy is an X-linked, adult-onset motor neuropathy caused by a progressive loss of lower motor neurons in the spinal cord and brainstem and sensory neurons in the dorsal root ganglia. Spinobulbar muscular atrophy, together with Huntington disease and fragile X syndrome, belongs to a growing group of neurodegenerative disorders caused by expansion of trinucleotide repeats. By targeting the CAG-expanded mRNA transcript with RNAi, Caplan et al11 suppressed polyglutamine cytotoxicity in cells. These studies provide a glimpse of the future potential of this RNAi approach to treat currently intractable neurological diseases. The siRNAs directed to disease alleles should not affect normal alleles, and thus should not disrupt function of normal cells. The major hurdle to applying RNAi to human disease therapy is an effective and safe method to deliver the siRNA to human patients. Recent work with lentiviral and adenoviral vectors suggests that future versions of these vectors may prove useful toward this goal.12
The technique of RNAi was discovered less than 2 years ago, yet its exploitation is proceeding by leaps and bounds. The results already obtained with the use of RNAi are exciting and point to a bright future for RNAi in determining the roles of orphan genes, in drug development, and, in the near future, toward direct uses to treat currently intractable human diseases.
Corresponding author and reprints: Dean P. Smith, MD, PhD, Department of Pharmacology and Center for Basic Science, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, TX 75390-9111 (e-mail: firstname.lastname@example.org).
Accepted for publication March 13, 2003.
Author contributions: Study concept and design, drafting of the manuscript, and critical revision of the manuscript for important intellectual content (Drs Kalidas and Smith); obtained funding (Dr Smith); administrative, technical, and material support (Dr Kalidas).
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