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The discovery of RNA interference in 1998 by Fire and coworkers provided an unanticipated new approach for studying
gene functions in many different cell types and species from various
kingdoms[1,2]. RNA interference (RNAi) is induced by
exogenously introduced double-stranded RNAs (dsRNAs), RNA viruses, transposons and endo-genous short dsRNAs,
and may be an ancient defense system against viral infections and other genetic
invaders[3]. The term RNA interference was
first introduced in 1998, after the discovery by Andrew Fire and collaborators that injection of long dsRNA into
Caenorhabditis elegans led to efficient and specific gene
silencing[1]. However, in contrast to the situation in invertebrates, introduction of
dsRNAs longer than 30 base pairs into mammalian somatic cells activates interferon responses, finally leading to a general
inhibition of gene expression. Nevertheless, the identification of small interfering RNAs (siRNAs), double-stranded RNAs
of 21 to 28 nucleotides in length, as intermediates of the RNAi process paved the way for applying RNAi in mammalian
cells[4]. Because of their short lengths, siRNAs only rarely induce interferon responses, but cause an efficient and
sequence-specific inhibition of gene expression. An increasing number of vector systems for the ectopic expression of small hairpin
RNAs (shRNAs), the intracellularly expressed cousins of siRNAs, complement improved chemical synthesis of siRNAs for
transient applications. All these advancements have allowed RNA interference to become a standard tool in molecular and
cellular biology within 3 years. Moreover, both siRNAs and shRNAs have been successfully applied
in vivo in animal models. Because of its high reproducibility, specificity and efficacy, in combination with rapid advances in the delivery of
RNAi-inducing molecules, RNAi holds great promise for the development of new therapeutic strategies to combat diseases
such as cancer[5,6].
Leukemia is a malignant disease of the hematopoietic system. Leukemic cells are characterized by impaired differentiation
and increased proliferation potential, leading to an expansion of the leukemic clone and the replacement of
normal hematopoiesis. In contrast to most types of solid tumors, more than 50% of all leukemia cases are associated with
distinct chromosomal changes, such as translocations or inversions. Moreover, leukemias tend to exhibit a higher genetic
stability than solid tumors. For these 2 reasons, molecularly defined approaches targeting tumor-specific genes such as
fusion genes may be more promising for leukemias than for solid tumors.
The leukemic fusion genes generated by chromosomal translocations are hallmarks of human leukemias. Prominent
examples are the BCR-ABL fusion gene, which is associated with chronic myeloid leukemia and acute lymphoblastic leukemia
(ALL), and AML1/MTG8, which is involved in acute myeloid leukemia (AML). Kinase fusion genes such as
BCR-ABL or TEL-PDGFbR directly disturb signal transduction pathways and provide proliferative and survival advantages. Chimeric
transcription factors such as AML1/MTG8,
PML-RARa or MLL-AF4 affect histone and DNA modifications and impair
differentiation and induce cell death. If such fusion genes still played central roles in the maintenance of leukemia, they could
be very promising siRNA targets for molecularly defined treatments, thereby complementing established treatment protocols
or new, small molecular drug-based strategies such as inhibition of histone deacetylation or DNA
methylation[7-9].
Here, we summarize a selection of such approaches to knockdown leukemic fusion proteins, and discuss the possible
applications of RNAi in the development of new therapies for leukemic diseases. A overview of several fusion
transcript-specific siRNAs is given in Table 1.
Short overview of the siRNA mechanism
Naturally occurring siRNAs are generated from long double-stranded RNAs by the RNase III-type enzyme Dicer. The
cleavage creates 5กฏ-phosphate and 3กฏ-hydroxyl termini and yields 21-28 long double stranded RNAs with 2 nucleotide-long
3กฏ-overhangs[10]. The siRNAs associate then with a multiprotein complex to form the RNA-induced silencing complex (RISC;
Figure 1). RISC is activated by unwinding the siRNA duplex and discarding one of the strands. Recent data suggest that a
Dicer-containing protein complex may also facilitate these 2
steps[11]. The remaining strand guides RISC to complementary
RNA sequences. The RISC component AGO2 cleaves the target sequence 11 nucleotides away from the 5กฏ-end of the siRNA,
leading to the rapid degradation and, thus, inactivation of the target
transcript[12,13]. Alternatively, particularly in the case of
imperfect homology between target sequence and guide strand, RISC may not cleave the target transcript, but may instead
inhibit its translation.
Translational interference without degradation of the transcript is also a feature of endogenously expressed micro RNAs
(miRNAs). The eminent roles of miRNAs in the regulation of gene expression and the consequences for cellular processes
such as differentiation or proliferation have only just recently come to the fore. In contrast to siRNAs, animal miRNAs have
an imperfect homology to their binding sequence, which seems to be responsible for the inability of miRNAs to trigger RNA
cleavage[14]. Nevertheless, despite differences in the mechanism (RNA degradation versus inhibition of translation), siRNAs
and miRNAs share protein components to form the corresponding nucleoprotein
complexes[15].
Both siRNA-mediated RNA degradation and miRNA-mediated inhibition of translation take place in the cyto-
plasm[16]. However, RISC does not seem to freely diffuse through the cytoplasm, but has been shown to be part of the
cytoplasmic P-bodies[17,18]. Thus, post-transcriptional gene silencing takes place in defined structures, like many other
essential cellular processes.
siRNA design and delivery exvivo
The efficiency and specificity of an siRNA is dependent on both siRNA and target site properties. One important point
to consider is the choice of the guide strand by RISC. Strand selection is controlled by the thermodynamic stability of the
siRNA termini. The strand with the lower 5กฏ-terminal thermodynamic stability is more likely to stay with RISC. Furthermore,
an adenosine residue at position 11 of the antisense strand, the complementary position of the cleavage site, can be
advantageous for cleavage efficiency, as RISC prefers to cleave RNA to the 3กฏ side of a uridine. However, these rules are not
absolute, and siRNAs, which do not adhere to these rules, do not necessarily have inferior activity. Targeting the fusion site
of a leukemic fusion transcript may, depending on the sequences flanking this site, not allow these rules to be strictly obeyed.
Instead, a compromise sequence must be found, or, if this is not possible, "non-consensus" siRNAs can be tried. For
instance, we identified active siRNAs by scanning a leukemic fusion site with several siRNAs. With this approach, we
obtained active and specific siRNAs, which did not follow any of the rules mentioned here (Figure
2)[19].
Transient transfections of siRNA duplexes into mammalian cells can be performed with various commercially available
cationic lipid formulations, or with
electroporation[20,21]. The advantage of such a transient approach is the lack of cell
adaptation to reduced target protein levels. Furthermore, genes essential for cell proliferation can be targeted by transient
siRNA approaches. However, there is no single transfection method that can be successfully applied to all cell types under
all experimental conditions. It is therefore important to optimize transfection conditions so that maximum gene silencing is
achieved. The following transfection parameters have been shown to affect transfection and gene silencing efficacy: cell
culture conditions, including cell density and medium composition; the type and amount of transfection agent; the quality
and quantity of siRNA; and the length of time that the cells are exposed to the siRNA. Differences have been reported in the
ability to transfect and silence gene expression between adherent and nonadherent cells. Post mitotic cells such as neurons
and muscle cells tend to be more difficult to transfect using liposomes compared with mitotic cells such as stem cells,
fibroblasts and tumor cells.
A major disadvantage of transfections using preformed siRNA lies directly in their transient nature: a long-lived protein
may not be affected by such an approach. To overcome this limitation, several siRNA expression systems based on plasmid
and viral vectors have been developed to achieve a sustained "knockdown"of the gene of
interest[22]. In most cases, the expression cassettes contain an RNA polymerase III-dependent promoter such as the U6 or the H1 promoters. The siRNAs
are expressed as hairpin structures known as short hairpin RNAs (shRNAs), which are processed by Dicer to the mature
siRNAs[23].
Because the application of plasmid vectors is frequently hampered by inefficient stable transfection rates and by
silencing of the shRNA genes, viral vector systems for the expression of shRNAs have been developed. In particular, lentiviral and
oncoretroviral systems have been demonstrated to be effective in most cell lines and primary cell types. Because lentiviruses
are able to infect non-cycling and post-mitotic cells, they are now widely used to establish a stable RNAi in, for example,
neuronal cells, stem cells and transgenic mouse
models[24].
Targeting kinases with siRNAs
BCR-ABL The vast majority of all chronic myeloid leukemia (CML) cases and a significant fraction of ALL cases are
associated with a chromosomal translocation t(9;22)(q34;q11), also known as the Philadelphia chromosome. This
translocation involves the reciprocal transfer of the 3กฏ Abelson proto-oncogene
(ABL1) sequence of chromosome 9 to variable
locations in the 3กฏ-breakpoint cluster region
(BCR) of chromosome 22. The resulting fusion
gene BCR-ABL is transcribed into 2 differently spliced chimeric mRNA encoding p210 or p190 BCR-ABL proteins. Both isoforms exhibit constitutively active
tyrosine kinase activity. The development of the
BCR-ABL rearrangement in hemopoietic stem cells (HSC) is the determining
event for the development of CML, leading to a cell clone with a competitive growth advantage over the normal stem
cells[25].
A major breakthrough in the treatment of BCR-ABL-
associated leukemia was the therapeutic application of the tyrosine kinase inhibitor imatinib mesylate (STI571, Gleevec).
Administration of this drug results in marked clinical and cytogenetic remissions. However, this compound is not perfectly
specific for BCR-ABL, but also inhibits the tyrosine kinase activities of ABL1, KIT and PDGFR. Moreover, and more
importantly, clinical resistance due to point mutations in the ABL kinase domain of BCR-ABL leading to an increasing
population of drug-resistant patients is frequently
observed[26,27]. These limitations necessitate the development of alternative strategies to complement existing treatment
protocols.
Different approaches have been used to deactivate the chimeric protein, such as single-stranded antisense
oligonucleotides, which, however, yielded conflicting results with respect to specificity and
functionality[28]. To overcome this, several groups used siRNAs to target
BCR-ABL mRNA as an alternative
approach[29-31]. Consistently, such siRNAs
decreased BCR-ABL expression without
affecting BCR and ABL1 expression. BCR-ABL depletion was associated with a
decreased leukemic proliferation and an increased extent of apoptosis. Despite the fact that cell lines were used in these
studies, they provide a basis for a possible siRNA application not only for functional analysis but also as a therapeutic tool.
The potential therapeutic value of a lentiviral gene transfer strategy was evaluated as an alternative to the exogenous
delivery of chemically synthesized
siRNAs[32,33]. The resultant stable expression of BCR-ABL shRNAs yielded a sustained
reduction of BCR-ABL, and, consequently, inhibited proliferation, decreased cell survival and compromised colony
formation of CD34+ CML cells. Withey
et al targeted BCR-ABL in primary chronic phase
CML[34]. The siRNA treatment inhibited the expansion of granulocyte-macrophage progenitors expressing the b3a2 variant of
BCR-ABL without having any obvious effects on cells expressing the b2a2 variant. This study underlines the role of
BCR-ABL in driving aberrant myeloid progenitor amplification.
Baba and colleagues targeted an siRNA against a downstream sequence in the
ABL portion instead of the fusion
site[35,36]. Thus, this siRNA has the potential to interfere with both
BCR-ABL and ABL1 expression. They demonstrated that this
siRNA markedly decreased target mRNA levels, consequently leading to the inhibition of protein tyrosine kinase activity and
suppression of cell proliferation. Microarray analysis revealed cross talk between siRNA-mediated suppression of the
BCR-ABL oncogene and
expression of several apoptotic/antiapoptotic and cell proliferation factors. Bartova and colleagues studied the dependence
of nuclear topography and expression of the BCR-
ABL fusion gene in the leukemic cell line K562 on the expression of nonfused
ABL1[37]. They showed that not only
BCR-ABL suppression but also inhibition of
ABL expression
induced downregulation of BCR-ABL and ABL1 proteins, which appeared to be sufficient for the stimulation of apoptosis.
Of the particular interest is the work of Wohlbold and collaborators, who demonstrated that combined treatment with
BCR-ABL siRNAs together with imatinib mesylate could overcome a partial imatinib mesylate
resistance[38]. In this case, decreasing the quantity of BCR-ABL protein using siRNA could antagonize the 2 major mechanisms of imatinib mesylate
resistance, namely overexpression of the protein and the occurrence of point mutations. Such applications of siRNAs as
supplementary therapy could be of clinical significance. The existence of several
BCR-ABL fusion transcript variants (eg b3a2 or b2a2) is a major limitation for the application of fusion site-specific siRNAs. However, Wohlbold
et al demonstrated in a follow-up study that all common BCR-ABL transcript variants could be successfully targeted with siRNAs that are homologous to the corresponding fusion sites.
TEL-PDGFbR The chromosomal translocation t(5;12)(q33;p13) associated with chronic myelomonocytic leukemia (CMML)
generates the TEL-PDGFbR fusion
gene[39]. Similar to BCR-ABL, the TEL-PDGFbR fusion protein exhibits a constitutively
active tyrosine kinase activity, which can be inhibited with imatinib mesylate or rapamycin. However, as with BCR-ABL, drug
resistance due to point mutations limits the therapeutic efficacy of these kinase inhibitors.
Chen et al applied an oncoretroviral transduction system for the delivery of
TEL-PDGFbR shRNAs[40]. The siRNAs were
transcribed using an H1-promoter-based short hairpin RNA expression system in a self-inactivating retroviral vector. The
TEL-PDGFbR siRNAs potently downmodulated TEL-PDGFbR, which affected signal transduction through PI3 kinase and
mammalian target of rapamycin (MTOR), and, consequently, inhibited the proliferation of
TEL-PDGFbR transformed cells. Furthermore,
TEL-PDGFbR depletion sensitized Ba/F3 cells expressing either wild-type
TEL-PDGFbR or a mutated, chemoresistant form to the small molecule inhibitors. Moreover, to evaluate the therapeutic efficacy of the siRNA
in vivo, TEL-PDGFbR-transformed Ba/F3 cells with or without coexpression of
the active siRNA were injected into the tail vein of
nude mice. Whereas Ba/F3 cells stably expressing
TEL-PDGFbR alone caused tumor development and death with a median
latency of 24 d after injection, expression of
TEL-PDGFbR siRNA resulted in significantly prolonged survival with a median latency of 41 d.
NPM-ALK Up to 75% of childhood and adolescent anaplastic large cell lymphomas (ALCL) carry the translocation
t(2;5)(p23;q35). In this case, the ubiquitously expressed nucleophosmin (NPM) is fused to the cytoplasmic tail of the anaplastic
lymphoma tyrosine kinase (ALK). The NPM-ALK fusion protein codes for a constitutively active tyrosine kinase, which is
necessary for cell transformation. This
kinase enhances factor-independent proliferation and inhibits apoptosis mostly via PI3K-Akt, Jak3/2-Stat3/5 and
phospholipase Cg signaling pathways. Ritter and collaborators designed and evaluated 3 chemically synthesized siRNAs for
downregulation of the NPM-ALK fusion mRNA[41]. The most potent of them reduced the levels of NPM-ALK mRNA
expression in SR786 ALCL cells by 50%-60%. However, repeated transfections were needed for significant reductions in the
protein level, probably due to the long half-life of the NPM-ALK protein. Nevertheless, the siRNAs in this case were
successfully used to dissect the signaling pathways employed by NPM-ALK. Similar to
TEL-PDGFbR, stable expression of an NPM-ALK shRNA sensitized
NPM-ALK-transformed Ba/F3 cells to rapamycin-induced cell death[40].
FLT3 Due to internal tandem duplication (ITD) within the juxtamembrane domain or due to point mutations, FMS-like
tyrosine kinase 3 (FLT3) is constitutively activated in 35% of all AML cases. In ALL carrying mixed lineage leukemia gene
(MLL) rearrangements, constitutive FLT3 activation is caused by overexpression of this kinase. Similar to other
studies[38], Walters et al examined the effects of siRNA-mediated
FLT3 suppression on the sensitivity towards a small-molecule FLT3
inhibitor, MLN518[42]. SiRNA-mediated FLT3 depletion diminished the phosphorylation of several downstream molecules,
thereby leading to the decreased viability of Ba/F3 and Molm-14 cells, which have internal tandem duplication of FLT3. The
combination of FLT3 siRNAs together with the specific FLT3 inhibitor MLN518 led to synergistic effects on cell proliferation
and apoptosis induction.
LYN As already described earlier, imatinib mesylate resistance due to BCR-ABL point mutations is a major problem in
CML therapy. Therefore, new, complementary approaches to interfere with BCR-ABL function such as siRNAs are currently
being intensively studied. Alternatively,
proteins other than BCR-ABL, which are also crucial for maintaining the leukemic
phenotype, may be promising targets. Members of the SRC kinase family such as SRC or LYN play a central role in
BCR-ABL-associated leukemogenesis. For that reason, Ptasznik
et al studied the effects of LYN depletion on the survival of imatinib
mesylate-resistant CML and ALL blast crisis
cells[43]. LYN siRNA reduced LYN protein in both normal hemopoietic cells and
BCR-ABL-expressing blasts by 80%-95%. Within 48 h, both siRNA-treated CML and ALL blasts underwent apoptosis,
whereas normal cells remained viable. Notably, ALL blasts seemed to be more affected by LYN depletion than CML blasts.
This increased dependence of BCR-ABL-positive leukemic blasts on LYN signaling provides the rationale for a
complementary treatment of imatinib mesylate-resistant CML blast crisis, particularly when lymphoid in nature.
Targeting transcriptional modulators with siRNAs
AML1/MTG8 The chromosomal translocation t (8;21)(q22;q22), which is the most frequent aberration associated with
10%-15% of all cases of AML, fuses the DNA-binding domain of the transcription factor RUNX1 (also called AML1 or
CBFa) to the almost complete open reading frame of RUNXT1 (also named MTG8, ETO or
CBFA2T1)[44]. Whereas RUNX1 is an essential transcription factor for definitive hemopoiesis, RUNXT1 or MTG8 is part of histone deacetylase-containing complexes.
Thus, the translocation converts a transcriptional modulator to a constitutive repressor of gene expression. The resulting
fusion protein AML1/MTG8 (AML1-ETO) inhibits myelopoiesis and supports the clonal expansion of hemopoietic stem
cells. Moreover, by directly binding to and sequestering transcription factors, such as SMAD3,
C/EBPa or vitamin D receptor, AML1/MTG8 interferes with signal transduction pathways controlling differentiation and
proliferation[45].
We examined the efficacy and specificity of siRNAs homologous to the fusion site of
AML1/MTG8 transcripts. Such siRNAs efficiently and specifically suppressed
AML1/MTG8 without interfering with
RUNX1 expression. AML1/MTG8 depletion led to severely impaired clonogenicity, a senescence-associated G1 cell cycle arrest, and an increased responsiveness to Tumor Growth Factor-b (TGFb)/vitamin D3-induced myelo-monocytic differentiation in t(8;21)-positive leukemic
cells[46]. The data imply a central role of AML1/MTG8 not only in the expansion of preleukemic progenitor cells, but also in the
maintenance of the leukemia. Moreover, AML1/MTG8 siRNAs are not only of value for the functional analysis of this fusion
gene, but may also be useful for an antileukemic therapy.
MLL-AF4 The chromosomal translocations involving human mixed lineage leukemia gene MLL located on chromosome
11 are associated with aggressive lymphoid and myeloid
leukemias[47]. MLL is the human trithorax homologue and stabilizes
unmethylated CpG islands. It is part of a several MDa large protein complex involved in the regulation of DNA methylation
and histone acetylation and methylation. MLL can be fused to more than 40 different partner genes, yielding a very diverse
collection of chimeric fusion
proteins[48]. The translocation partners do not share any unifying properties. Nevertheless, 2
different classes can be distinguished. One group of partners such as AF1 or GAS7 cause oligomerization of the
corresponding fusion protein[49], whereas a second group (eg AF4, AF9, ENL) might be part of a single multiprotein
complex[50,51]. The molecular mechanisms of MLL-associated leukemogenesis are currently being intensively studied. Despite the diversity of
the fusion proteins, characteristic features of MLL-associated leukemias are an inappropriate expression of a subset of
homeotic genes, such as MEIS1 and HOXA9,
and the overexpression of FMS-like tyrosine kinase 3
(FLT3)[52].
The translocation t(4;11) is associated with a very aggres-sive and therapy-resistant acute lymphoblastic leukemia in
infants and with therapy-related secondary
leukemias[53,54]. Recently, we showed that transient inhibition
of MLL-AF4 expression with small interfering RNAs impaired the proliferation and clonogenicity of the t(4;11)-positive human leukemic
cell lines SEM and RS4;11[19]. Reduction of MLL-AF4 levels induced apoptosis associated with caspase-3 activation and
diminished BCL-XL expression. Suppression of
MLL-AF4 was paralleled by a decreased expression of the homeotic genes
HOXA7, HOXA9, and MEIS1. Moreover, MLL-AF4 depletion inhibited expression of the hemopoietic stem cell marker
CD133, indicating hemopoietic differentiation. Finally, transfection of leukemic cells with MLL-AF4 siRNAs reduced
leukemia-associated morbidity and mortality in xenotran-splanted
SCID mice, suggesting that MLL-AF4 depletion negatively
affects leukemia-initiating cells. Our findings demonstrate that
MLL-AF4 is important for leukemic clonogenicity and
engraftment of this highly aggressive leukemia.
To date, the presence of MLL rearrangements are indicative of a poor clinical outcome, partially due to the lack of specific
pharmacological inhibitors[55]. Therefore, the siRNA approach might be an option for developing new strategies for the
treatment of these aggressive leukemias.
Challenges and promises
The examples discussed in the previous sections strongly suggest that siRNA-mediated oncogene suppression may
become a promising option in antileukemic therapy. However, there are major obstacles to overcome, such as specificity,
induction of an interferon response, emergence of escape mutations, or inefficient systemic siRNA delivery
in vivo. Only if these challenges can be successfully addressed will siRNA technology fulfill its promises in cancer therapy.
siRNA specificity and possible off-target effects should be given particular consideration. Like all other antisense
molecules, siRNAs tolerate mismatches to a certain extent, possibly compromising their sequence
specificity[56]. Even if mismatches prevent RISC-mediated cleavage of unintended
target transcripts, protein expression may still be
affected[57]. Furthermore, siRNAs or shRNAs may compete with endogenously expressed miRNAs for RISC components, thereby
affecting miRNA-regulated gene expression and cellular processes such as
differentiation[58,59]. Finally, both chemically
synthesized siRNAs as well as intracellularly expressed shRNAs may induce a limited interferon response both in cell culture and
in vivo[60]. This induction proceeds via Toll-like receptors and/or protein kinase R, but the parameters responsible are just
currently becoming clear[61-63]. One possible method of controlling siRNA- triggered interferon response is to examine the
induction of classical interferon response genes such as STAT1 and OAS1. Furthermore, ectopic expression of an
siRNA-insensitive variant of the target protein would be a suitable control for the possible side-effects of siRNAs. However, such
rescue experiments are sometimes difficult to perform. For instance, ectopic expression of AML1/MTG8 or MLL-AF4 inhibits
cell proliferation and induces cell death in many different cell types. An alternative might be the sequence-specific inhibition
of different variants of the fusion gene. For instance, we targeted 2 different MLL-AF4 variants with specific
siRNAs[19]. Each siRNA only inhibited the proliferation of that cell line expressing the perfectly complementary variant, but had no effect
on the other cell line. Because both siRNAs are functional in their corresponding cell lines, such an experimental setting is
equivalent to a rescue experiment.
Another point of concern is the ability of siRNAs to remain functional in the context of possible escape mutations.
However, we and others have demonstrated that different fusion gene variants can be successfully targeted with
siRNAs[19,38]. Thus, it may be possible to counteract an escape mutation by simply adapting the siRNA sequence.
The most challenging problem for the therapeutic application of siRNAs is the efficient delivery of siRNA into leukemic
tissues, including leukemic stem cells. To date, several techniques have been examined to obtain systemic siRNA delivery in
mouse models[64]. However, approaches such as high-pressure, high-volume intravenous injection of synthetic siRNAs, the
so-called hydrodynamic delivery, are of limited clinical use because of the severe side
effects[65].
Recently, the pharmacokinetics and efficacies of chemically modified siRNAs containing a 5กฏ-cholesterol moiety on the
sense strand in combination with limited phosphoro-thioate and 2กฏ-methoxy modification were examined in a mouse model.
Intravenous injection resulted in the silencing of ApoB mRNA in liver and jejunum, decreased plasma levels of ApoB protein,
and reduced total cholesterol[66]. Alterna-tively, unmodified siRNAs have been successfully delivered by using vehicles
such as polyethylenimine, atelocollagen, cationic lipids or neutral
liposomes[67-71]. In all these cases, intravenous injection of
the corresponding vehicle-siRNA mixture inhibited gene expression and tumor growth in mouse models.
The major goal for RNAi approaches for cancer therapy is the selective elimination of tumor cells without damaging
normal cells. For that, if targeting a non-mutated gene, siRNAs should be selectively delivered to the transformed cells.
Alternatively, siRNAs may target genes that are exclusively expressed in cancer cells, and that are crucially involved their
growth or survival (Figure 3). The latter option is preferable when a fusion gene is involved in tumorigenesis. The examples
discussed in this review demonstrate the suitability and effectiveness of this approach both in cell culture and, after siRNA
delivery ex vivo, also in murine model systems. Given the rapid progress being made in the field of therapeutic RNA
interference, antileukemic siRNAs may be not too far away from complementing existing therapeutic protocols to treat
leukemia.
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