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J Biomed Biotechnol. 2006; 2006: 71659. Published online 2006 May 18. doi: 10.1155/JBB/2006/71659. | PMCID: PMC1559929 |
Delivery Systems for the Direct Application of siRNAs to Induce
RNA Interference (RNAi) In Vivo Achim Aigner* Department of Pharmacology and Toxicology, Philipps-University Marburg, Karl-v.-Frisch-Strasse 1, 35033 Marburg, Germany Received January 14, 2006; Accepted February 27, 2006. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work is properly cited. RNA interference (RNAi) is a powerful method for specific gene
silencing which may also lead to promising novel therapeutic
strategies. It is mediated through small interfering RNAs (siRNAs)
which sequence-specifically trigger the cleavage and subsequent
degradation of their target mRNA. One critical factor is the
ability to deliver intact siRNAs into target cells/organs in vivo.
This review highlights the mechanism of RNAi and the guidelines
for the design of optimal siRNAs. It gives an overview of studies
based on the systemic or local application of naked siRNAs or the
use of various nonviral siRNA delivery systems. One promising
avenue is the the complexation of siRNAs with the polyethylenimine
(PEI), which efficiently stabilizes siRNAs and, upon systemic
administration, leads to the delivery of the intact siRNAs into
different organs. The antitumorigenic effects of
PEI/siRNA-mediated in vivo gene-targeting of tumor-relevant
proteins like in mouse tumor xenograft models are described. Altered expression levels of certain genes play a
pivotal role in several pathological conditions. For example,
in many cancers the upregulation of
certain growth factors or growth factor receptors, or the
deregulation of intracellular signal transduction pathways,
represents key elements in the process of malignant transformation
and progression of normal cells towards tumor cells leading to
uncontrolled proliferation and decreased apoptosis. Since these
processes may result in the direct, autocrine stimulation of the
tumor cell itself as well as the paracrine stimulation of other
cells, including the stimulation of tumor-angiogenesis, many novel
therapeutic strategies focus on the reversal of this effect, that
is, the inhibition of these proteins or the downregulation of
their expression. Likewise, several other diseases have been
firmly linked to the (over-)expression of endogenous wildtype or
mutated genes. Taken together, in addition to strategies based on
the inhibition of target proteins, for example, by low molecular
weight inhibitors or inhibitory antibodies, this opens an avenue
to gene-targeting approaches aiming at decreased expression of the
respective gene. The first method to be introduced for the specific inhibition of
gene expression was the use of antisense oligonucleotides in the
late 1970s [ 1,
2]. Upon their introduction into a cell,
antisense ODNs are able to hybridize to their target RNA leading
to the degradation of the RNA-DNA hybrid double strands through
RNAase H, to the inhibition of the translation of the target
mRNA due to a steric or conformational obstacle for protein
translation and/or to the inhibition of correct splicing. In the
early 1980s, the discovery of ribozymes, that is, catalytically
active RNAs which are able to sequence-specifically cleave a
target mRNA, further expanded gene-targeting strategies
[ 3– 5]. Subsequently, both methods were extensively
studied and further developed with regard to the optimization of targeting
efficacies and antisense-ODN/ribozyme delivery strategies in vitro and in vivo. Most recently, another naturally occurring biological strategy for
gene silencing has been discovered and termed RNA interference
(RNAi). Since RNAi represents a particularly powerful method for
specific gene silencing and is able to provide the relatively easy
ablation of the expression of any given target gene, it is now
commonly used as a tool in biological and biomedical research.
This includes the RNAi-mediated targeting in vitro and in vivo for
functional studies of various genes whose expression is known to
be upregulated as well as the development of novel therapeutic
approaches based on gene targeting. RNAi is an evolutionarily conserved, sequence-specific,
posttranscriptional gene silencing phenomenon. It is triggered by
double-stranded RNA molecules as described first in C elegans by
Fire et al [ 6] who then introduced the name RNA
interference. These findings also explained earlier observations
in petunias which turned white rather than purple upon the
introduction of the “purple gene” in form of dsRNA
[ 7], and
on gene silencing by antisense oligonucleotides as well as by
sense oligonucleotides in C elegans [ 8].
Subsequent studies demonstrated that RNAi, while described under different names
(posttranscriptional gene silencing (PTGS), cosuppression,
quelling), is present in most eukaryotic organisms with the
response to dsRNA, however, being more complicated in higher organisms. RNAi relies on a multistep intracellular pathway which can be
roughly divided into two phases, that is, the initiation phase and
the effector phase. In the initiation phase, double-stranded RNA
molecules from endogenous or exogenous origin present in the cell
are processed through the cleavage activity of a ribonuclease
III-type protein [ 9– 12] into short 21–23 nucleotide
fragments termed siRNAs. These effector siRNAs, which contain a
symmetric 2 nt overhang at the 3′-end as well as a
5′-phosphate and a 3′-hydroxy group, are then in the effector
phase incorporated into a nuclease-containing multiprotein
complex called RISC (RNA-induced silencing complex)
[ 13].
Several structural and biochemical studies have shed light on the
processing of double-stranded RNA and the formation of the RISC
complex (see, eg, [ 14] for a recent review). Through
unwinding of the siRNA duplex by an RNA helicase activity
[ 15], this complex becomes activated
with the single-stranded siRNA guiding the RISC complex to its complementary target RNA.
Upon the binding of the siRNA through hybridization to its target
mRNA, the RISC complex catalyses the endonucleolytical cleavage of
the mRNA strand within the target site, which, due to the
generation of unprotected RNA ends, results in the rapid
degradation of the mRNA molecule. With the RISC complex being
recovered for further binding and cleavage cycles, the whole
process translates into a net reduction of the specific mRNA
levels and hence into the decreased expression of the
corresponding gene. For an overview of the RNAi pathway, see
. While from this mechanism it becomes obvious that siRNA molecules
complementary to the target mRNA and thus being able to serve as a guide
sequence for the RISC complex play a pivotal role in this process, they need
not be derived from long double-stranded precursor molecules. Rather,
omitting the initiation phase, they can be delivered directly into the
target cell (, upper right arrow). Several studies have led to the development of guidelines for the
generation of siRNAs which are optimal in terms of efficacy and
specificity [ 12,
16]. This includes the initial definition of
the preferable length (19–25 bp) combined with a low G/C
content in the range between 36% and 52% and the requirement
of symmetric 2 nt overhangs at the 3′-end
[ 16– 18].
Later studies on synthetic siRNA molecules, however, revealed an
up to 100-fold higher targeting efficacy in the case of even
longer duplexes (25–30 nucleotides) which act as a substrate for
Dicer and which therefore allow the direct incorporation of the
newly produced siRNAs into the RISC complex [ 19].
As to be expected, intramolecular foldback structures which can result
from internal repeats or palindrome sequences decrease the numbers
of functional siRNA molecules with silencing capability
[ 20].
Additional silencing-enhancing criteria include an A in position 3
and a G at position 13 of the sense strand, the absence of a C or
G at position 19 and, most importantly, a U in position 10 of the
sense strand. Since nucleotides 10–11 represent the site of the
RISC-mediated cleavage of the target mRNA, this indicates that
RISC is comparable to most other endonucleases in preferentially
cleaving 3′ of U rather than any other nucleotide
[ 20, 21].
Furthermore, it was shown more generally that the thermodynamic
flexibility of the positions 15–19 of the sense strand
correlates with the silencing efficacy and that the presence of at
least one A/U base pair in this region improves siRNA-mediated
silencing efficacy due to a decreased internal stability of its
3′-end [ 20]. Still, different siRNA sequences may display differing efficacies, which
suggest additional still unknown criteria for optimal siRNA selection and
emphasize the influence of target mRNA accessibility. In fact, several
studies also correlate the siRNA efficacy with the mRNA secondary structure
[ 18, 22– 27]. In conclusion, apart from the selection criteria defined above, the
individual screening of different siRNAs for highly efficient and specific
duplexes, or the pooling of multiple siRNAs, is the most effective
approach to increase siRNA-mediated targeting efficacy. For the design of effective siRNAs, several algorithms on publicly
accessible web sites are available (see [ 28]
for review). To reduce the risk of nonspecific (“off-target”)
effects of the siRNAs, a homology search of the targeting sequence against a gene database is
necessary and already incorporated in some of these web sites. Nevertheless,
it has also been shown that siRNAs may cross-react with targets of limited
sequence similarity when regions of partial sequence identity between the
target mRNA and the siRNA exist. In fact, in some cases regions comprising
of only 11–15 contiguous nucleotides of sequence identity were sufficient to
induce gene silencing [ 29]. The prediction of these
off-target activities is difficult so far. An additional mechanism that may lead to nonspecific effects in
vivo relies on the interferon system [ 30– 33]
which is induced when double-stranded RNA molecules enter a cell activating
a multi-component signalling complex. This effect is particularly
true for long dsRNA molecules and essentially prevents them from
being used as inducers of RNA interference in mammalian systems.
The development of synthetic siRNAs [ 10,
12, 33,
34] largely
circumvents this problem since they seem to be too small. However,
some synthetic siRNAs do induce components of the interferon
system which seems to be dependent on their sequence
[ 31, 32,
35]
as well as, in the case of in vitro transcribed siRNAs, on the 5′
initiating triphosphate [ 36]. Thus,
strategies to avoid as far as possible the unwanted interferon
response upon application of siRNAs in vivo will include a design
of siRNAs without known interferon-stimulating sequences, the use
of the lowest possible siRNA dose to still achieve the desired
effect and optimized siRNA delivery methods. OLIGONUCLEOTIDE DELIVERY SYSTEMS Based on the known mechanisms of antisense technology, ribozyme-targeting
or RNAi, small oligonucleotides or plasmid-based expression vectors can be used
to specifically downregulate the expression of a given gene of interest or
of pathological relevance in vitro. In principle, this also applies to the
in vivo situation leading to novel, potentially relevant therapeutic approaches. For the delivery of therapeutic nucleic acids, viral vectors have
been used which have the advantage of high transfection efficacy
due to the inherent ability of viruses to transport genetic
material into cells. On the other hand, however, viral systems
show a limited loading capacity regarding that the genetic material are
rather difficult to produce in a larger scale and, most
importantly, pose severe safety risks due to their oncogenic
potential and their inflammatory and immunogenic effects which
prevent them from repeated administration [ 37– 40]. In the light of these problems, concerns, and limitations, nonviral systems
have emerged as a promising alternative for gene delivery. Main requirements
are the protection of their nucleic acid “load” as well as their efficient
uptake into the target cells with subsequent release of the DNA or RNA
molecules and, if necessary, their transfer into the nucleus. Several
strategies can be distinguished, mainly lipofection and polyfection relying
on cationic lipids or polymers, respectively (see, eg,
[ 41– 43]). The efficient protection against enzymatic or nonenzymatic
degradation is particularly important for RNA molecules including
siRNAs. In fact, while the therapeutic potential of siRNAs for the
treatment of various diseases is in principle very promising,
limitations of transfer vectors may turn out to be rate-limiting
in the development of RNAi-based therapeutic strategies. One
approach to solve this problem is the use of DNA expression
plasmids which encode palindromic hairpin loops with the desired
sequence. Upon transcription and folding of the RNA, the
double-stranded short hairpin RNAs (shRNAs) are recognized by
Dicer and cleaved into the desired siRNAs. Additionally, an in
vitro method has been described recently which is based on the
expression of shRNAs in E coli and their delivery via bacterial
invasion [ 44]. While
all these different DNA-based systems
offer the advantage of siRNA expression with a longer duration and
a probably higher level of gene silencing, they still rely on
(viral or nonviral) delivery of DNA molecules and again raise
safety issues in vivo. Hence, the direct delivery of siRNAs
molecules, derived from in vitro transcription or chemically
synthesized, offers advantages over DNA-based strategies and may
be preferable for in vivo therapeutic use. In the last years, a large body of studies has been published
which describe different strategies for the systemic or local
application of siRNAs in vivo. Tables 1– 3
give an overview. The probably largest number of papers focuses
the use of unmodified siRNAs ( Table 1) whose
administration is often performed IV by hydrodynamic transfection
(high pressure tail vein injection). While this method is widely
used and in some cases led to efficient target gene inhibition in
the liver and, to a lesser extent, in lung, spleen, pancreas, and
kidney, it may suffer from certain technical and practical
limitations at least in a therapeutical setting since it relies on
the rapid IV injection of a comparably large volume (>= 1
ml/mouse/injection, in theory equivalent to a ~ 3 l
IV bolus injection in man). Alternative strategies for the
application of naked siRNAs include various delivery routes which,
however, often provide an only local administration or rely on an
administration at least close to the target tissue or target
organ, thus restricting the number of target organs which may not
be relevant for certain diseases. It should also be noted that
several studies described here and below use rather large amounts
of siRNAs and that upon intravenous injection of siRNAs the liver
is the primary site of siRNA uptake. As an alternative approach
for the application of siRNAs in vivo, their delivery by
liposomes/cationic lipids has been described. For liposome-based
siRNA formulations, a wide variety of modes of application
allowing local or systemic delivery has been used
( Table 2). Finally, several other strategies for local
or systemic siRNA administration have been explored, including
chemical modifications of siRNA molecules, electropulsation,
polyamine, or other basic complexes, atelocollagen, virosomes, and
certain protein preparations ( Table 3). | Table 1Studies based on the direct application of siRNAs to
induce RNAi in vivo: administration of unmodified siRNAs. |
| Table 3Studies based on the direct application of siRNAs to induce RNAi in
vivo: other strategies of siRNA administration. |
| Table 2Studies based on the direct application of siRNAs to induce RNAi in
vivo: administration of siRNAs based on liposomes/cationic lipids. |
An alternative approach relies on the complexation of unmodified siRNA
molecules with a cationic polymer, polyethylenimine (PEI). POLYETHYLENIMINES: FROM DNA TRANSFECTION
TO siRNA DELIVERY IN VITRO AND IN VIVO Polyethylenimines (PEIs) are synthetic polymers available in
branched or linear forms (, upper panels) and
in a broad range of molecular weights from < 1000 Da to
> 1000 kd. Commercial PEI preparations, although labelled
with a defined molecular weight, consist of PEI molecules with a
broad molecular weight distribution [ 45– 47].
PEIs possess a high cationic charge density due to a protonable amino group in
every third position [ 48,
49]. Since no quarternary amino
groups are present, the cationic charges are generated by
protonation of the amino groups and hence are dependent on the pH
in the environment (eg, 20% at pH 7.4, see
[ 50] for
review). Due to its ability to condense and compact the DNA into
complexes, which form small colloidal particles allowing efficient
cellular uptake through endocytosis, PEI has been introduced as a
potent DNA transfection reagent in a variety of cell lines and in
animals for DNA delivery (for review, see [ 51,
52] and
references therein). In fact, in several studies PEI has been
shown to be able to deliver large DNA molecules such as
2.3 Mb yeast artificial chromosomes (YACs)
[ 53] as well
as plasmids or small oligonucleotides [ 48,
54– 56] into
mammalian cells in vitro and in vivo. The N/P ratio, which
indicates the ratio of the nitrogen atoms of PEI to DNA phosphates
in the complex and thus describes the amount of PEI used for
complex formation independent of its molecular weight, influences
the efficiency of DNA delivery. A positive net charge of the
complexes, resulting from high N/P ratios, inhibits due to
electrostatic repulsion their aggregation and improves their
solubility in aqueous solutions as well as their interaction with
the negatively charged extracellular matrix components and thus
their cellular uptake [ 57]. Additionally,
the strong buffer capacity, described by the “proton sponge
hypothesis” which
postulates enhanced transgene delivery by cationic polymer-DNA
complexes (polyplexes) containing H + buffering polyamines due
to enhanced endosomal Cl −
accumulation and osmotic
swelling/lysis [ 48],
seems to be responsible for the fact
that PEI-based delivery does not require endosome disruptive
agents for lysosomal escape. This tight condensation of the DNA
molecules as well as the buffering capacity of PEI in certain
cellular compartments like endosomes and lysosomes also protects
DNA from degradation [ 48,
49, 58,
59]. PEIs have been
successfully used for nonviral gene delivery in vitro and in vivo.
While initial publications showed increased transfection
efficacies when using high molecular weight PEIs
[ 45], more
recent studies demonstrated the advantages of certain low
molecular weight PEIs [ 47,
60,
61]. The higher transfection
efficacy of low molecular weight PEIs may be due to a more
efficient uptake of the resulting PEI/DNA complexes, a better
intracellular release of the DNA and/or lower in vitro
cytotoxicity as compared to high molecular weight PEI
[ 60– 63]
. In fact, a decrease in the molecular weight of
the PEI leads to an increase in complex size which may be
favourable at least for in vitro use [ 64,
65]. On the other
hand, other PEIs with very low molecular weight (< 2 kd)
display little or no transfection efficacy even at very high N/P
ratios which may be attributed to the fact that a decrease in the
molecular weight of PEI has been shown to translate into an
increasingly lower ability to form small complexes [ 63].
Therefore, low molecular weight PEIs require higher N/P ratios
for optimal transfection efficacies as compared to higher
molecular weight PEIs since higher N/P ratios lead to an increase
in compaction with reduced complex sizes and a reduced tendency of
the complexes to aggregate due to hydrophobic interactions
[ 61, 63,
64]. Nevertheless, while several parameters
have been extensively studied, some precise determinants for transfection
efficacy remain to be elucidated (see [ 50,
66] for review).
Also, the mechanism of the cytotoxic effects of PEI complexes is
only poorly understood. It may rely on the formation of large
aggregates in the range of up to 2 μm which, when formed on
the cell surface, impairs membrane functions finally leading to
cell necrosis [ 60]. Clearly,
there is a trend towards low
molecular weight PEIs as rather nontoxic delivery reagents in
vitro and in vivo, which combine high biocompatibility and reduced
side-effects thus also allowing to employ larger PEI/DNA complex
amounts without significant cytotoxicity. More recently, the use of polyethylenimines has been extended
towards the complexation and delivery of RNA molecules, especially
small RNA molecules like 37 nt all-RNA ribozymes
[ 67– 69]
and siRNAs [ 70] (). While
chemically unmodified RNA molecules are very instable and prone to
rapid degradation, the PEI complexation has been shown to lead to
an almost complete protection against enzymatic or
nonenzymatic degradation. In fact, PEI-complexed siRNAs, which are
[ 32P]-labeled for better detection, remain intact in vitro
for several hours even in the presence of RNase A or fetal calf
serum at 37 °C, while non-complexed siRNAs are rapidly
degraded (). This indicates that
siRNA molecules are efficiently condensed and thus fully covered
and protected by PEI. Indeed, the analysis of PEI/siRNA complexes
by atomic force microscopy showed the absence of free siRNAs or
siRNA molecule ends and thus confirms these findings regarding an
efficient complexation (Grzelinski et al, submitted). However,
while the complex stability seems to be sufficient for siRNA
protection with all PEIs tested (Werth et al, in press; Aigner et
al, unpublished data), several of these complexes do not show any
targeting efficacy at all. In fact, only when using certain
polyethylenimines, PEI/siRNA complexes are efficiently delivered
into target cells in vitro, where siRNAs are released and display
bioactivity (Figures and
). In general and
as seen before for PEI/DNA complexes (see above), the transfection
efficacy is dependent on the PEI used, also indicating that the
siRNA targeting efficiency mainly depends on the endocytotic
uptake of the complex and/or its intracellular
decomposition rather than on the in vitro complex stability. Good
results were obtained with commercially available JetPEI [ 70]
while the in vivo JetPEI from the same supplier showed only poor
siRNA delivery efficacies [ 71]. Likewise, a novel low
molecular weight PEI based on the fractionation of a commercially
available polyethylenimine demonstrates high siRNA protection and
delivery efficacies in vitro (Werth et al, in press). Under
certain conditions, the PEI/RNA (siRNA or ribozyme)
complexes retain their physical stability and biological
activity also after lyophilization ([ 72] and Werth et al, in
press). Although the PEI transfection is only transient, data from
our lab show that PEI/siRNA effects are stable for at least 7 days
(Urban-Klein and Aigner, unpublished results). Finally, another
study has explored the use of siRNA nanoplexes comprising of PEI
that is PEGylated with an RGD peptide ligand attached at the
distal end of the PEI. Again, siRNA nanoplexes protect siRNAs
against serum degradation and show in vitro activity [ 73]. The ultimate goal is the application of siRNAs in vivo which has
been explored in some studies in different mouse models. Ge et al
showed that PEI-complexed siRNAs targeting conserved regions of
influenza virus genes are able to prevent and treat influenza
virus infection in mice. Upon IV injection, PEI promoted the
delivery of siRNAs into the lungs where, either given before or
after virus infection, siRNA reduced influenza virus production in
the lungs [ 74]. Most biological effects of the systemic application of
PEI-complexed siRNAs, however, have been determined in different
mouse tumor models and by targeting different proteins which have
been shown previously to be tumor-relevant. This includes the
epidermal growth factor receptor HER-2 (c-erbB-2/neu), the growth
factor pleiotrophin (PTN), and vascular endothelial growth factor
(VEGF) and its receptor (VEGF R2), and the fibroblast growth
factor-binding protein FGF-BP. The in vivo administration of PEI complexed, but not of naked
siRNAs, through IP or subcutaneous injection resulted in the detection of
intact siRNAs even hours after injection
(). Radiolabeled siRNA molecules were found
in several organs including subcutaneous tumors, muscle liver,
kidney and, to a smaller extent, lung and brain. It is important
to note that the siRNAs were actually internalized by the tissues
as indicated by the fact that blood was negative for siRNAs
(). Overexpression of the HER-2 receptor has been observed in a wide variety of
human cancers and cancer cell lines. Since HER-2 displays strong cell
growth-stimulating and antiapoptotic effects especially through heterodimer
formation with other members of the EGFR family, its overexpression has been
established as a negative prognostic factor and linked to a more aggressive
malignant behaviour of tumors (eg, [ 75]).
Consequently, HER-2 qualifies as an attractive target molecule for antitumoral treatment
strategies including anti-HER-2 antibodies, low molecular weight inhibitors,
or HER-2-specific gene-targeting approaches. In fact, the relevance of HER-2
(over-)expression in tumor growth has been established in several in vitro
HER-2 targeting studies including the use of ribozymes
[ 76, 78,
79]
or siRNAs [ 80,
81]. It was
demonstrated that HER-2 reduction in vitro leads, among others, to the
inhibition of cell proliferation and increased apoptosis. The systemic treatment of athymic nude mice bearing subcutaneous SKOV-3
ovarian carcinoma tumor xenografts through IP injection of PEI-complexed
HER-2-specific siRNAs led to marked antitumoral effects as seen by a
significant reduction tumor growth ()
[ 70].
PEI-complexed nonspecific siRNAs or HER-2-specific, naked siRNAs had no
effects. This was paralleled by the detection of intact HER-2-specific
siRNAs in the tumors of the specific treatment group already 30 min after
administration and for at least 4 h, and by the downregulation of HER-2 on
mRNA and protein levels [ 70]. Another receptor, VEGF R2, was targeted in a study employing self-assembling
nanoparticles based on siRNAs complexed PEI which is PEGylated with an RGD
peptide ligand attached at the distal end of PEG. While the PEGylation
allows steric stabilization and reduces nonspecific interactions of the
complexes, the RGD motif provided tumor selectivity due to their ability to
target integrins expressed on activated endothelial cells in the tumor
vasculature. Upon IV administration into mice bearing subcutaneous N2A
neuroblastoma tumor xenografts, a selective tumor uptake and a VEGF R2
downregulation were observed, resulting in decreased tumor growth and tumor
angiogenesis [ 73]. The receptor ligand, VEGF, is a mitogenic and angiogenic growth factor
stimulating tumor growth and angiogenesis in several tumors including
prostate carcinoma. Thus, it may represent attractive target molecule for
RNAi-based gene-targeting strategies also bearing in mind the double
antitumoral effect due to reduction of tumor cell proliferation as well as
tumor angiogenesis. The subcutaneous or intraperitoneal injection of
VEGF-specific siRNAs complexed with a novel PEI obtained through
fractionation of a commercially available PEI (Werth et al, in press)
resulted in the reduction of tumor growth due to decreased VEGF expression
levels (Höbel and Aigner, unpublished results). The same was true for
PEI/siRNA-mediated targeting of FGF-BP (Dai and Aigner, unpublished
results), which has been established previously as “rate-limiting” for tumor
growth and angiogenesis in several tumors ([ 82,
83], see [ 84] for review). Finally, PEI/siRNA-mediated targeting of pleiotrophin (PTN) exerted strong
antitumoral effects. PTN is a secreted growth factor which shows mitogenic,
chemotactic, angiogenic and transforming activity
[ 85– 93]
and which is markedly upregulated in several human tumors including cancer
of the breast, testis, prostate, pancreas, and lung as well as in melanomas,
meningiomas, neuroblastomas, and glioblastomas. The in vivo treatment of nude
mice through systemic subcutaneous or IP application of PEI-complexed PTN siRNAs
led to the delivery of intact siRNAs into subcutaneous tumor xenografts and a
significant inhibition of tumor growth. Likewise, in a clinically more
relevant orthotopic mouse glioblastoma model with U87 cells growing
intracranially, the injection of PEI-complexed PTN siRNAs into the CNS
exerted antitumoral effects. This establishes, also in a complex and
relevant orthotopic tumor model, the potential of PEI/siRNA-mediated PTN
gene targeting as a novel therapeutic option in GBM, and further extends the
modes of delivery of PEI/siRNA complexes intrathecal strategies as employed
in the therapy of glioblastomas with antisense oligonucleotides. Only a few years after their discovery, siRNAs are catching up with
ribozymes and antisense oligonucleotides as efficient tools for
gene targeting in vitro and, more recently, also in vivo. This includes the
exploration of their potential as therapeutics which will lead to the
development of siRNA-based therapeutic strategies. Their ultimate success,
however, will strongly depend on the development of powerful and feasible
siRNA delivery strategies which need to address several issues including the
stability/stabilization of siRNA molecules while preserving their efficacy
and maintaining their gene-silencing activity, an efficient delivery into
the target organ(s) as well as a sufficiently long siRNA half life in the
organism and particularly in the target organ. Thus, siRNA delivery
strategies must provide siRNA protection and transfection efficacy, the
absence of toxic and nonspecific effects, they must be efficacious also
when using small amounts of siRNAs and must be applicable in various
treatment regimens and in various diseases even when this requires to
overcome biological barriers after their administration to reach their
target tissue or target organ. The research done on DNA-based gene delivery,
ribozyme-targeting, and antisense technology will facilitate this process
since it already provides a basis of established technologies. This is also
true for the complexation of siRNAs with polyethylenimine, which may
represent a promising avenue for siRNA applications in vivo. This may
eventually lead to novel therapeutic strategies. The work of A. Aigner is supported by the Deutsche
Forschungsgemeinschaft (AI 24/5-1)
and by the Deutsche Krebshilfe. The author would like to apologize
to the authors whose primary works have not been cited due to length
considerations. ABBREVIATIONS | | dsRNA, | double-stranded RNA, | | | FGF-BP, | fibroblast growth factor-binding protein, | | | GFP, | green fluorescent protein, | | | HER-2, | human epidermal growth factor receptor-2, | | | IP, | intraperitoneal, | | | ODN, | oligodeoxynucleotide, | | | PEI, | polyethylenimine, | | | PTN, | pleiotrophin, | | | RISC, | RNA-induced silencing complex, | | | RNAi, | RNA interference, | | | siRNA, | small interfering RNA, | | | shRNA, | short hairpin RNA. |
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