Small Interfering RNAs:
`
`i o n a r y T o o l
` G e n e
`A R e v o l u t
` o f
`s
`t h e A n a l y s
`i o n a n d G e n e T h e
`
`f o r
`t
`F u n c
`
`r a p y
`
`i
`
`The ability of double-stranded RNA
`(dsRNA) to silence the expression of
`genes has been the focus of many
`studies in C. elegans and D.
`melanogaster. More recent research has
`looked for evidence of RNAi-mediated
`gene suppression in other model
`organisms. Now there is excitement
`that RNAi-based methodologies will
`allow for the rapid assessment and
`validation of proteins as potential drug
`targets. Additionally, we might now be
`standing on the edge of fundamentally
`new approaches to gene therapy as
`conducted through RNAi-mediated
`suppression of mutated genes.
`
`RNA interference (RNAi) represents an evolutionarily
`
`conserved cellular defense for controlling the expression
`
`of foreign genes in most eukaryotes including humans. RNAi is
`
`triggered by double-stranded RNA (dsRNA) and causes
`
`sequence-specific mRNA degradation of single-stranded target
`
`RNAs homologous in response to dsRNA. The mediators of
`
`mRNA degradation are small interfering RNA duplexes (siRNAs),
`
`which are produced from long dsRNA by enzymatic cleavage in
`
`the cell. siRNAs are approximately twenty-one nucleotides in
`
`length, and have a base-paired structure characterized by two-
`
`nucleotide 3'-overhangs. Chemically synthesized siRNAs have
`
`become powerful reagents for genome-wide analysis of
`
`mammalian gene function in cultured somatic cells. Beyond
`
`their value for validation of gene function, siRNAs also hold
`
`great potential as gene-specific therapeutic agents.
`
`Thomas Tuschl1 and Arndt Borkhardt2
`1Department of Cellular Biochemistry, Max-Planck-Institute
`for Biophysical Chemistry, D-37077 Goettingen, Germany
`2Children’s University Hospital, Pediatric Hematology and
`Oncology, 35392 Giessen, Germany
`
`158
`
`Alnylam Exh. 1055
`
`

`

`A Role for siRNAs in Genetic Therapy
`
`INTRODUCTION
`
`When viruses infect eukaryotic cells, or when transposons and
`transgenes are randomly integrated into host genomes, dsRNA is
`frequently produced from the foreign genes. Most eukaryotes,
`including humans, possess an innate cellular immune surveillance
`system that specifically responds to the presence of dsRNA and
`activates processes that act post-transcriptionally to silence the
`expression of the interloping genes (1–4). This mechanism is now
`commonly referred to as RNA interference or RNAi (5). During
`RNAi, long transcripts of dsRNA are rapidly processed into small
`interfering RNAs (siRNAs), which represent RNA duplexes of
`specific length and structure that finally guide sequence-specific
`degradation of mRNAs homologous in sequence to the siRNAs (6,
`7). The sequencing of the human genome has created an urgent
`need to ascertain efficiently the function of novel genes and to
`validate targets for drug discovery. Indeed, the rapid translation of
`the genomic DNA sequence information into therapeutic strategies
`for many common maladies—particularly infectious,
`cardiovascular, neoplastic, and neurological diseases—would be
`highly desirable. siRNAs may be the best tools for target validation
`in biomedical research today, because of their exquisite specificity,
`efficiency and endurance of gene-specific silencing. siRNAs are
`probably also suitable for the design of novel gene-specific
`therapeutics by directly targeting the mRNAs of disease-related
`genes.
`The transfection of siRNAs into animal cells results in the
`potent, long-lasting post-transcriptional silencing of specific genes
`(8, 9). siRNA-mediated gene silencing is particularly useful in
`somatic mammalian cells, because these cells mount an additional,
`sequence-nonspecific innate immune response (i.e., responding
`with interferon-mediated defenses) when exposed to dsRNA
`greater than thirty base pairs, therefore prohibiting the application
`of longer dsRNAs (10). siRNAs are extraordinarily effective at
`lowering the amounts of targeted RNA—and by extension
`proteins—frequently to undetectable levels. The silencing effect is
`long lasting, typically several days, and extraordinarily specific,
`because one nucleotide mismatch between the target RNA and the
`central region of the siRNA is frequently sufficient to prevent
`silencing (6, 7, 11, 12). siRNAs can be rapidly synthesized and are
`now broadly available for the analyses of gene function in cultured
`mammalian cells. Similar to antisense oligonucleotide technology
`(13), the use of siRNAs also holds great promise for the application
`of gene-specific therapies in treating acute diseases such as viral
`infection, cancer, and, perhaps, acute inflammation.
`
`THE MECHANISM OF DSRNA INTERFERENCE
`
`A schematic illustration shows the mechanism of RNAi (Figure 1).
`The key enzyme required for processing of long dsRNAs to siRNA
`duplexes is the RNase III enzyme Dicer, which was characterized
`in extracts prepared from insect cells, C. elegans embryos, and
`
`dsRNA
`
`siRNAs
`
`siRNP/RIS
`
`target RNA
`cleavage
`
`7mG
`
`product release
`
`7mG
`
`Dicer RNase III
`
`Ago2, ?
`
`…
`
`+7mG
`
`AAAAA
`
`Primed RdRP
`polymerization
`
`AAAAA
`
`Unprimed RdRP
`polymerization
`
`AAAAA
`
`Figure 1. A model for RNA interference. dsRNA is processed to 21- to
`23-nt siRNA duplexes by Dicer RNase III and possible other dsRNA-
`binding factors. The siRNA duplexes are incorporated into a siRNA-
`containing ribonucleoprotein complex (siRNP) (21) becoming the RNA-
`induced silencing complex (RISC) endonuclease, which targets
`homologous mRNAs for degradation. AGO2 and yet to be characterized
`proteins are thought to be required for RISC formation. The RISC mediates
`sequence-specific target RNA degradation. In plants and nematodes,
`targeted RNAs might also function as templates for double-strand RNA
`synthesis giving rise to transitive RNAi (24, 80–83), either through siRNA-
`primed dsRNA synthesis or through unprimed synthesis from aberrant
`RNA (which could represent the cleaved target RNA). In mammals or in
`fruitfly, however, RdRP genes have yet been identified, and the major
`mechanism of siRNA action is believed to be endonucleolytic target RNA
`cleavage guided by siRNA-protein complexes (RISC).
`
`mouse cells (14–16). Dicer contains an N-terminal RNA helicase
`domain, a Piwi, Argonaute, Zwille/Pinhead (PAZ) domain (17), two
`RNase III domains, and a C-terminal dsRNA-binding motif. The
`PAZ domain is also present in Argonaute proteins, whose genes
`represent a poorly characterized family present in dsRNA-
`responsive organisms. Argonaute1 (AGO1) and Argonaute2
`(AGO2), two of the five Argonaute proteins of D. melanogaster,
`appear to be important for forming the mRNA-degrading
`sequence-specific endonuclease complex, also referred to as the
`RNA-induced silencing complex (RISC) (18, 19). Dicer and AGO2
`appear to interact in D. melanogaster Schneider 2 (S2) cells,
`probably through their PAZ domains; however, RISC and Dicer
`activity are separable, and RISC is unable to process dsRNA to
`siRNAs, suggesting that Dicer is not a component of RISC (18, 20).
`Possibly, the interaction between Dicer and AGO2 facilitates the
`incorporation of siRNA into RISC (20). The endonucleolytic
`subunit of RISC remains to be identified.
`siRNA duplexes produced by the action of Dicer contain 5'-
`
`June 2002
`Volume 2, Issue 3
`
`159
`
`

`

`Review
`
`TABLE 1. SIRNA-MEDIATED SILENCING IN CELL LINES
`Tissue origin
`Reference
`Cell line
`human epidermoid carcinoma
`A-431
`(23)
`human lung carcinoma
`A549
`(72)
`human B-precursor leukemia
`BV173
`(77)
`human papilloma virus negative cervical carcinoma
`C-33A
`(46)
`human Burkitt’s lymphoma
`CA46
`(77)
`human colon epithelial cells
`Caco2
`(61)
`Chinese hamster ovary
`CHO
`(23)
`African green monkey kidney
`COS-7
`(9)
`rat fibroblast
`F5
`(32)
`human nonsmall cell lung carcinoma
`H1299
`(46)
`human keratinocyte cell
`HaCaT
`(12)
`human embryonic kidney
`HEK 293
`(9)
`human papilloma virus positive cervical carcinoma
`HeLa
`(9)
`human hepatocellular carcinoma
`Hep3B
`(64)
`human umbilical vein endothelial cells
`HUVEC
`(54)
`human diploid fibroblast
`IMR-90
`(41)
`human chronic myelogenous leukemia, blast crisis
`K562
`(77)
`human T-cell lymphoma
`Karpas 299
`(77)
`human breast cancer
`MCF-7
`(84)
`MDA-MB-468 human breast cancer
`(84)
`MV-411
`human acute monocytic leukemia
`(77)
`NIH/3T3
`mouse fibroblast
`(9)
`P19
`mouse embryonic carcinoma
`(42)
`SD1
`human acute lymphoblastic leukemia
`(77)
`SKBR3
`human breast cancer
`(23)
`U2OS
`human osteogenic sarcoma cell
`(59)
`
`siRNAs are generated that may cleave the mRNA
`outside of the region targeted by the ancestral
`dsRNA. Although this appears to have important
`implications for RNAi-based analysis of gene
`function because silencing may spread between
`genes that share highly homologous sequences,
`phenotypic analysis of a large set of silenced genes
`in C. elegans suggests that transitive RNAi between
`naturally occurring homologous gene sequences is
`probably of no major concern (25, 26). It was also
`proposed that siRNAs might prime novel dsRNA
`synthesis; however, it should be pointed out that
`siRNAs, in comparison to longer dsRNAs, are
`extremely poor initiators of gene silencing in C.
`elegans (27, 28). Biochemical evidence for RdRP
`activity in D. melanogaster was recently reported
`(29), although genes encoding classical RdRP
`activity appear to be lacking from the D.
`melanogaster genome. Despite the beauty of the
`suggested model in D. melanogaster, which
`hypothesizes that siRNAs function as primers for
`target-RNA-dependent dsRNA synthesis, thus
`leading to amplification of the silencing signal (29),
`biochemical evidence for the spreading of gene
`silencing outside of regions targeted by dsRNAs has
`not been observed in other model systems (6, 12,
`23, 30, 31). Rather, the predominant pathway of
`gene silencing appears to be siRNA-mediated target
`mRNA degradation by RISC formation, which may
`also act catalytically. Similar to the situation in D. melanogaster,
`genes encoding RdRPs have not been identified in mammals.
`Therefore it is important to remember that the mechanisms of
`silencing differ between different species.
`
`APPLICATION OF SIRNAS IN SOMATIC MAMMALIAN CELLS
`
`siRNAs have brought reverse genetics to mammalian cultured
`cells, and have made large-scale functional genomic analysis a
`realistic possibility (32). Standard cell lines provide starting points
`for mammalian functional screens because siRNAs can be
`effectively delivered by electroporation or cationic liposome-
`mediated transfection (11, 23). For small scale-applications, the
`microinjection of siRNAs may represent an alternative method.
`Technical problems that result from low transfection efficiencies
`may be partially overcome by using cell sorting protocols, such as
`after the transfection of siRNAs together with sorting markers such
`as GFP-expression plasmids. Alternatively, siRNAs that target cell
`surface marker proteins may be co-transfected, and the reduced
`expression of the co-targeted cell surface marker could then be
`used to identify specific cell populations by cell sorting.
`An obvious prerequisite for the application of siRNAs for
`validation and therapeutic applications is the need for functional
`
`phosphates and free 3'-hydroxyl groups. The central base-paired
`region is flanked by two-to-three nucleotides of single-stranded 3'-
`overhangs (6). The 5'-phosphate termini of siRNAs is essential for
`guiding mRNA degradation (21). Nevertheless, for their practical
`application in gene targeting experiments, siRNAs may be used
`without 5'-phosphate termini because a kinase activity in the cell
`rapidly phosphorylates the 5' ends of synthetic siRNA duplexes (9,
`21, 22). Under certain circumstances (e.g., injection experiments
`in D. melanogaster), 5'-phosphorylated siRNA duplexes may have
`slightly enhanced properties as compared to 5'-hydroxyl siRNAs
`(22). In gene targeting experiments using human HeLa cells, no
`differences in siRNA-mediated “knockdown” of gene expression
`were observed, as a function of 5'-phosphorylation (23).
`Furthermore, a cell line that is unable to utilize synthetic 5'-
`hydroxyl siRNAs for RNAi has not been encountered (for cell lines
`supporting RNAi see Table 1).
`In C. elegans, introduction of approximately 300 bp dsRNA
`corresponding to a segment of the targeted gene may also give rise
`to the phenomenon of transitive RNAi (24). Transitive RNAi is
`characterized by the spreading of silencing outside of the region
`targeted by the initiator dsRNA. Presumably, targeted mRNA
`serves as template for RNA-dependent RNA polymerase (RdRP)
`and forms new dsRNA that is processed by Dicer. Thus, secondary
`
`160
`
`

`

`A
`
`7mG
`
`NA
`
`(N19)
`
`NN
`
`AAAAA
`
`5´
`
`3´
`
`TT
`
`TT
`
`3´
`
`sense
`
`5´
`
`antisense
`
`target
`mRNA
`
`siRNA
`
`B
`
`H1 RNA
`promoter
`
`T5
`
`5´
`
`3´UU
`
`Figure 2. Methods for the delivery of siRNAs to somatic mammalian
`cells. (A) Synthetic 21-nt siRNA duplex prepared by chemical synthesis
`(23) aligned to a target mRNA. Target regions are selected such that
`siRNA sequences may contain uridine residues in the 2-nt overhangs.
`Uridine residues in the 2-nt 3'-overhang can be replaced by 2'-
`deoxythymidine without loss of activity, which significantly reduces
`costs of RNA synthesis and may also enhance nuclease resistance of
`siRNA duplexes when applied to mammalian cell (7). (B) Plasmid-based
`expression of short hairpin loops which give rise to siRNAs in vivo (11).
`The polymerase III promoter of H1 RNA (human RNase P RNA) drives
`the transcription of a nineteen-base-pair/nine-nucleotide-loop RNA
`hairpin. The transcription is terminated by the encounter of a
`polythymidine tract (T5) after the incorporation of two to three uridine
`residues encoded by the T5 element. Northern blot analysis showed that
`the hairpin RNAs were processed to siRNAs.
`
`RNAi machinery within the targeted cells or tissue to bind to
`siRNAs and mediate mRNA degradation. In order to assay for the
`activity of this ribonucleoprotein complex in cells, a reporter assay
`was developed (9, 23). Plasmids coding for firefly and sea-pansy
`luciferase are transfected together with control or target-specific
`(i.e., luciferase) siRNAs into cells, and the relative luminescence of
`target versus control luciferase activity is measured.
`siRNAs have been used to identify cytoskeletal proteins that
`are essential for cell growth (32). Even the targeting of non-
`essential genes resulted in cellular phenotypes that were identical
`to phenotypes previously observed in cells derived from transgenic
`knockout mice (32), illustrating the value of siRNA methodology
`for the analysis of mammalian gene function.
`In certain situations, several-hundred-base-pair long dsRNA
`represents an alternative to siRNAs. Long dsRNA effectively
`silences genes expressed in insect cells (18, 33–35) and in
`embryonic mammalian cells that have not yet established the
`interferon system (15, 36–39). However, undifferentiated cells,
`
`A Role for siRNAs in Genetic Therapy
`
`Chromosome 22
`
`BCR
`
`13
`12
`11.2
`
`11.2
`12
`13
`
`22
`
`BCR
`
`24
`23
`21
`13
`12
`
`12
`13
`21
`
`22
`31
`32
`33
`34
`
`Chromosome 9
`
`ABL
`
`9
`
`ABL
`
`p210 kD
`chimeric
`BCR/ABL
`oncoprotein
`
`Fusion site
`
`BCR
`
`ABL
`
`siRNA-meduated
`targeting of the
`fusion site
`
`Chronic myelogenous leukemia
`
`Figure 3. Scheme of the translocation t(9;22) in leukemia. The BCR-
`ABL fusion mRNA provides a leukemia-specific target that can be cleaved
`by siRNAs.
`
`such as embryonic stem (ES) cells or P19 teratocarcinoma cells,
`are difficult to work with because these progenitor cells are often
`poorly transfectable, making cell sorting prior to phenotypic
`analysis necessary (15).
`Until recently, the application of siRNAs in somatic cells was
`restricted to the delivery of chemically or enzymatically
`synthesized siRNAs (9, 40–42) (Figure 2A), but methods for
`intracellular expression of small RNA molecules have now been
`developed. Endogenous delivery is possible by inserting DNA
`templates for siRNAs into RNA polymerase III (pol III)
`transcription units, which are based on the sequences of the
`natural transcription units of the small nuclear RNA U6 or the
`human RNase P RNA H1. Two approaches are available for
`expressing siRNAs: 1) The sense and antisense strands constituting
`the siRNA duplex are transcribed from individual promoters
`(42–44), or 2) siRNAs are expressed as fold-back stem-loop
`structures that give rise to siRNAs after intracellular processing by
`Dicer (11, 41, 42, 45, 46) (Figure 2B). The transfection of cells
`with plasmids that encode siRNAs, therefore, represents an
`alternative to direct siRNA transfection. Stable expression of
`siRNAs may facilitate certain applications such as the functional
`characterization of non-essential gene products in synthetic
`lethality screens or the construction of combinatorial libraries
`useful for loss-of-function screening in microarray assays (47). The
`insertion of siRNA expression cassettes into (retro)viral vectors will
`also enable the targeting of primary cells refractory to transfection
`or electroporation of plasmid DNA.
`The function of several genes in cultured somatic mammalian
`cells have been analysed using siRNAs. The human vacuolar
`sorting protein Tsg101 was thus identified as essential for HIV-1
`but not MLV budding (48). The reintroduction of a Tsg101-
`
`June 2002
`Volume 2, Issue 3
`
`161
`
`

`

`Review
`
`expressing plasmid that encoded Tsg101 mRNA with silent
`mutations at the siRNA-targeting site restored the HIV-1 budding
`defect. siRNA-mediated depletion of endogenous targets and the
`re-introduction of siRNA-resistant rescue constructs (30) will
`become important for the analysis of protein function much like
`the complementation analyses used in traditional yeast genetic
`research. In other instances, siRNAs were applied for studying the
`role of proteins involved in DNA damage response and cell cycle
`control (11, 49–53), general cell metabolism (54–56), signaling
`(57–59), cytoskeleton and its rearrangement during mitosis (32,
`44, 60), membrane trafficking (61, 62), transcription (63), and
`DNA methylation (64). These various examples reported by
`independent investigators illustrate the robustness of the siRNA
`gene silencing technology.
`However, variations do exist in the efficiency of siRNAs to
`target the same genes (12, 23, 32). Our experience in targeting
`many different genes suggests that, on average, between seventy to
`ninety percent of randomly chosen siRNAs are able to reduce
`target gene expression by more than eighty percent. Some genes,
`encoding extremely abundant and extremely stable proteins (e.g.,
`vimentin), may be more difficult to silence, and the probability for
`finding efficacious siRNAs may be lower (23, 32). Nonetheless,
`difficulty in depleting the most abundant proteins does not appear
`to compromise the value of this new transient gene silencing
`technology.
`
`SIRNAS AS NOVEL THERAPEUTIC PLATFORM TECHNOLOGY
`
`(RSV) (72), a negative strand RNA virus that causes sometimes
`severe respiratory disease, especially in neonates and infants. They
`also demonstrated that siRNAs do not induce interferon-mediated
`responses, by showing the absence of phosphorylation of the
`translation factor eIF-2␣. Although mRNAs transcribed from the
`viral genome were effectively silenced and viral replication was
`inhibited, it was not possible to cleave the viral genomic or
`antigenomic RNA because of its chromatin-like condensed
`structure. Lee et al. recently reported effective siRNA-mediated
`degradation of HIV-1 rev transcripts in a cell assay by co-
`transfection of proviral DNA and siRNA expression vectors, thus
`raising the possibility that siRNAs may become useful to treat HIV
`infection (43).
`In leukemias and lymphomas—the most frequent cancers in
`childhood—oncogene activation frequently occurs through
`reciprocal chromosomal translocations. These translocations lead
`to juxtaposition of gene segments normally found on different
`chromosomes, and the creation of a composite gene. The
`prototype of such a translocation is the generation of the
`Philadelphia chromosome by the translocation of the long arms of
`chromosomes 9 and 22 [t(9;22)] in patients with chronic
`myelogenous leukemia and acute lymphoblastic leukemia (73).
`The translocation fuses the BCR gene from chromosome 22 and
`ABL gene from chromosome 9, creating an oncogenic BCR-ABL
`hybrid gene (Figure 3) (74). The BCR-ABL fusion protein has
`dramatically increased the tyrosine kinase activity, as compared to
`that of the normal ABL protein, leading to aberrant
`phosphorylation of several downstream molecules. The kinase
`activities of both BCR-ABL and ABL can be inhibited by a specific
`tyrosine kinase inhibitor, STI 571 (Imatinib®), which is now used
`in the effective treatment of BCR-ABL-positive leukemia (75, 76).
`RNAi was also used to target the BCR-ABL mRNA, and this
`approach was compared to that of STI 571–mediated cell killing in
`
`TABLE 2. SELECTION OF POSSIBLE TARGETS FOR TUMOR THERAPY BY SIRNAS
`Aberration
`Tumors
`
`siRNAs are highly sequence-specific reagents and discriminate
`between single mismatched target RNA sequences (7, 11), and may
`represent a new avenue for gene therapy. The expression of
`mRNAs coding for mutated proteins, which give rise to dominant
`genetic disorders and neoplastic growth, might be decreased or
`blocked completely by specific
`siRNAs.
`With respect to targeting viral
`gene products expressed in virus-
`infected cells, it is possible that
`infectious mammalian viruses
`could express inhibitors of RNAi
`similar to those identified in plant
`and insect viruses (65–71).
`Because viral inhibitors of the
`mammalian RNAi machinery have
`not yet been described, it seems
`feasible that the application of
`siRNAs could extend our
`understanding of viral protein
`function and viral life cycle. Bitko
`and Barik successfully used
`siRNAs to silence genes expressed
`from respiratory syncytial virus
`
`Genes or
`Fusion Genes
`RAS
`
`c-MYC, N-MYC
`
`ERBB
`ERBB2
`MLL fusion genes
`BCR-ABL
`TEL-AML1
`EWS-FLI1
`TLS-FUS
`PAX3-FKHR
`BCL-2
`
`AML1-ETO
`
`162
`
`Point mutations
`
`Overexpression, translocation,
`Point mutation, amplification
`overexpression
`Overexpression
`Translocation
`Translocation
`Translocation
`Translocation
`Translocation
`Translocation
`Overexpression,
`translocation
`Translocation
`
`Pancreatic carcinoma, chronic leukemia,
`colon carcinoma, lung cancers
`Burkitt’s lymphoma, neuroblastoma
`
`Breast cancer
`Breast cancer
`Acute leukemias
`Acute and chronic leukemia
`Childhood acute leukemia
`Ewing sarcoma
`Myxoid liposarcoma
`Alvelolar rhabodomyosarcoma
`Lung cancers, Non-Hodgkin
`lymphoma, prostate cancer
`Acute leukemia
`
`

`

`A Role for siRNAs in Genetic Therapy
`
`a cell culture model. The siRNA treatment readily reduced the
`expression of BCR-ABL mRNA, followed by a reduction of BCR-
`ABL oncoprotein, leading to apotosis in leukemic cells (77).
`siRNA-based BCR-ABL silencing may become important
`considering that some patients develop drug resistance against STI
`571 (e.g., by genomic amplification of BCR-ABL, increased
`expression of BCR-ABL mRNA or point mutation in the ABL
`gene). Alternative therapies, perhaps applied in combination with
`inhibitors such as STI 571, may help to overcome problems of
`such drug resistance.
`The combined effort of many laboratories worldwide has led
`to the molecular clarification of numerous chromosomal
`translocations through the successful cloning of the genes adjacent
`to the chromosomal breakpoint regions. Silencing of these tumor-
`specific, chimeric mRNAs by siRNAs might become an effective
`fusion gene-specific tumor therapy. The extraordinary sequence
`specificity of the RNAi mechanism may also allow for the targeting
`of individual polymorphic alleles expressed in loss-of-
`heterozygosity tumor cells, as well as targeting point-mutated
`transcripts of transforming oncogenes such as Ras. Finally, the
`decrease of overexpressed apoptosis inhibitors such as Bcl-2 and c-
`Myc might also be beneficial for cancer therapy. A list of possible
`targets for siRNA-mediated therapy in human malignancies is
`shown in Table 2.
`With respect to future medical applications, siRNAs were
`recently directed against a mutated mRNA associated with the
`spinobulbular muscular atrophy (SBMA) in tissue culture (78).
`SBMA, together with Huntington Disease, belongs to a growing
`group of neurodegenerative disorders caused by the expansion of
`trinucleotide repeats (79). Targeting the CAG-expanded mRNA
`transcript with dsRNA may be an attractive alternative to
`commonly used therapeutic strategies that, beyond symptomatic
`treatment, mainly focus on the inhibition of the toxic effects of the
`polyglutamine protein. Caplen et al. (78) successfully decreased
`the expression of mutated transcripts of the androgen receptor in
`human kidney 293T cells that were transfected with a plasmid
`encoding the expanded-CAG androgen receptor mutant. Most
`importantly, the authors achieved a rescue of the polyglutamine-
`induced cytotoxicity in cells treated with dsRNA molecules. Even
`though the study observed RNAi in transfected cells in vitro rather
`
`than in a more physiologically relevant context, the approach
`provides proof-of-principle, and underlines the remarkably broad
`potency and sequence-specificity of RNAi-mediated gene therapy.
`Whether the RNAi pathway is functionally active in various
`neuronal cells irrespective of their state of differentiation remains
`to be shown.
`The delivery of siRNAs to the proper sites of therapy remains
`problematic. This is especially true for their delivery to primary
`cells, because such cells often do not tolerate treatment with
`liposome transfection reagents. Chemical modification of siRNAs,
`such as changing the lipophilicity of the molecule may be
`considered—for example, phosphorothioate modifications present
`in antisense oligodeoxynucleotides, or the attachment of lipophilic
`residues at the 3'-termini of the siRNA duplex. Delivery of siRNAs
`into organisms might be achieved with methods previously
`developed for the application of antisense oligonucleotides or
`nuclease-resistant ribozymes. Such methods consist of the
`injection of naked or liposome-encapsulated molecules. Studies
`that inform us about the possibility of exploiting RNAi in various
`cell types, tissues, and organs are urgently needed. Without doubt,
`these experiments will be performed in the near future in
`academic as well as industrial settings.
`
`CONCLUSIONS
`
`The pace with which siRNAs revolutionize the analysis of
`mammalian gene function is astounding, considering that siRNA-
`mediated gene silencing was only introduced last year (9, 78).
`siRNAs are poised to facilitate the genome-wide systematic analysis
`of gene function in cultured cells, and may soon become a
`valuable tool for target validation beyond in vitro tissue culture.
`siRNAs may yet provide a solution for gene-specific drug
`development, especially before highly specific small-molecule
`inhibitors become available.
`
`Acknowledgments
`We acknowledge Sayda Elbashir, Jens Harborth, Klaus Weber and
`Ulrike Krämer for critical comments on the manuscript. Our own
`studies were made possible by grants from the Deutsche
`Forschungsgemeinschaft.
`
`References
`
`1. Hammond, S.M., Caudy, A.A., and
`Hannon, G.J. Post-transcriptional gene
`silencing by double-stranded RNA.
`Nat. Rev. Genet. 2, 110–119 (2001).
`
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