New approaches for cancer treatment: antitumor drugs based on gene-targeted nucleic acids.

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44 | ActA nAturAe | № 2 2009
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New Approaches
for Cancer Treatment:
Antitumor Drugs Based on
Gene-Targeted Nucleic Acids
O.A. Patutina, N.L. Mironova, V.V. Vlassov, M.A. Zenkova*
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian
Academy of Sciences, Novosibirsk
*E-mail: marzen@niboch.nsc.ru
ABSTRACT currently, the main way to fight cancer is still chemotherapy. this method of treatment is at the height of its capacity, so, setting aside the need for
further improvements in traditional treatments for neoplasia, it is vital to develop now approaches toward treating malignant tumors.
this paper reviews innovational experimental approaches to treating malignant malformations based on the use of gene-targeted drugs, such as antisense oli-
gonucleotides (asOn), small interfering rnA (sirnA), ribozymes, and D nAzymes, which can all inhibit oncogene expression. the target genes for these drugs
are thoroughly characterized, and the main results from pre-clinical and first-step clinical trials of these drugs are presented. It is shown that the gene-targeted
oligonucleotides show considerable variations in their effect on tumor tissue, depending on the target gene in question. the effects range from slowing and stop-
ping the proliferation of tumor cells to suppressing their invasive capabilities. Despite their similarity, not all the antisense drugs targeting the same region of the
mrnA of the target-gene were equally effective. the result is determined by the combination of the drug type used and the region of the target-gene mrnA
that it complements.
Keywords: cancer therapy, antisense oligonucleotides, ribozymes, DnAzymes, small interfering rnA
Abbreviations: antisense oligonucleotides (asOn), small interfering rnA (sirnA), rnA interference (rnAi)
INTRODuCTION
At the current stage of modern medicine, one of the most
important projects is to increase the effectiveness of cancer
treatment by searching for and developing new therapies
and improving traditional therapeutic approaches. A com-
bination of surgery, radio- and chemotherapy is still the
golden standard for cancer treatment, and these approaches
have led to an 8-fold increase in patient survival over the
last 30 years. the negative features of surgery-only treat-
ment are recurrent tumors, the spread of metastases, and
the formation of unresectable malignant malformations.
this forces doctors to use radio- and chemotherapy. Alas,
even this combination of powerful cancer therapies often
doesn’t bring positive results. therefore, despite the unde-
niable achievements of modern oncology, increasing the ef-
fectiveness of cancer treatment is of utmost importance.
During the last several decades, complex chemotherapy
has become the main approach for treating cancer patients.
Its use however is limited, despite the fact that it increas -
es survival rates by 30% to 90%, depending on the type of
malformation. the main hindrances are systemic toxicity,
nonselective action (the effect is not specifically targeted
towards tumor tissue), and the emergence of drug-resistant
tumor cell clones.
recent discoveries have provided scientists with detailed
knowledge of the molecular processes underlying carcino-
genesis, tumor invasiveness, angiogenesis, and metastasis, as
well as other processes, such as tumor suppression, growth
control, apoptosis, and immune response. these data have
led to the development of a new generation of chemothera-
peutic drugs, such as Gleevec (aka Glivec or Imatinib mesy-
late), Mabtherа (aka rituximab), etc., which have a highly
selective effect on their cellular target. It is well known that
creating a new drug takes about 10–20 years of research,
and improving its selectiveness increases its cost mani-
fold. currently, chemotherapy as a high-dose active attack
aimed at tumor cells is at the limit of its ability. Despite the
achieved level of patient survival (for certain cancer types it
has made a 10-fold increase in the last 20 years), there is still
a 10% to 70% proportion of patients who, for a number of
reasons, do not react to treatment. therefore, the creation of
new methods of therapy is a relevant problem at this time.
Among the drugs which are currently being developed,
gene-targeted drugs are of considerable interest. the pos-
sibility of inhibiting a gene’s expression was first discovered
in the ground-breaking research of n.I. Grineva and her col-
leages [1–3] and was studied further in order to regulate the
expression of genes involved in carcinogenesis using anti-
sense [4] and gene-targeted oligonucleotides [5]. currently,
the main lines of inquiry into gene-targeted cancer therapy
are strategies to suppress oncogene overexpression, restore
the expression of tumor suppressing genes, boost the activ-
ity of the immune system, suppress angiogenesis and metas-
tasis, and initiate tumor self-destruction.
this paper reviews the new experimental approaches to
cancer treatment based on gene-targeted oligonucleotides,

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№ 2 2009 | ActA nAturAe | 45
asON
a
RNase H
mRNA – target
mRNA – target
translation
blocking
mRNA –
destruction
ribosome
b
which are currently being used in experiments on cell cul-
tures and laboratory animals. Some of these drugs are also
in various stages of clinical trial.
hOW gENE-TARgETED NuCLEIC ACIDS WORk
Antitumor drugs based on nucleic acids are highly specific
tools which allow the gene expression to be regulated, and
they have been attracting the attention of scientists as pos-
sible regulators of carcinogenesis at the molecular level. the
suppression of several genes whose anomalously high ex-
pression is associated with neoplastic transformation can
be achieved by nucleic acid-based drugs, such as antisense
oligonucleotides (asO n), small interfering rnAs (si rnA),
ribozymes, and DnAzymes. Generally speaking, the mech-
anism of gene suppression by these drugs is the comple-
mentary binding of oligonucleotides to their mrnA target,
which causes the target m rnA molecule to be destroyed or
blocks its translation.
AsOn are synthetic single-strand DnA, 15–20 nucle-
otides long, and they can form a complementary complex
with the target m rnA sequence [6]. Protein synthesis is
suppressed by asO n due to the fact that the m rnA target
is degraded by the intracellular rnase H, which identi -
fies the hybrid DnA/rnA complex (Fig. 1a), or due to a
block of translation, since the formation of a hybrid com-
plex hampers the ribosome’s movement on the m rnA
strand (Fig. 1b) [7]. recently discovered asO n can block
the transfer of spliced mrnA from the nucleus to the cy-
toplasm; other asOn can block a splicing site in pre-mr-
nA and thus cause the expression of an alternate protein
product. [8, 9].
the ability of asOn to selectively suppress the production
of a protein was demonstrated by Zamechnik and Stephen-
son in 1978 [4]. they showed that a 13-base oligonucleotide
complementary to the 3′ -terminal sequence of the rnA of
the rouse sarcoma virus inhibits viral replication in vitro.
this study led to the research of asOn as a potential thera-
peutic method in cancer and viral infection treatment, as
well as treatments for inflammatory processes, blood dis-
eases, and cardio-vascular conditions [10–14].
Since it is known that naturally occurring oligodeoxy-
ribonucleotides are rapidly degraded by nucleases in vivo
and in cell cultures, several chemical modifications are in-
corporated into the asOn structure to increase their stabil-
ity [11]. these modifications increase not only asOn stabil-
ity against nucleases, but also their biological effectiveness,
hybridization efficiency, and cellular uptake. Among the
most notable antisense oligonucleotide derivates are thi-
ophosphate oligonucleotides, in which one of the oxygen
atoms not incorporated in the phosphodiester bond is sub-
stituted for a sulphur atom [11,15]. thiophosphate asO n
are resistant to nucleases, highly soluble, effective in hy-
dridization, and form a heteroduplex with mrnA, which is
targeted by rnase H [11]. One drawback of thiophosphate
asOn is their high affinity to a range of proteins [16, 17].
Second-generation asOn carried an alkyl residue in the
ribose 2’-position: 2’-О -methyl and 2’-О -methoxyethyl oli-
goribonucleotides were effective in blocking mrnA trans -
lation, but they did not activate the degradation of the
mrnA/asOn heteroduplex by rnase H. [18]. Later nielsen
and co-authors substituted the sugar-phosphate backbone
of the nucleic acid to an n-(2’-aminoethyl)-glycine poly-
amide structure [19], which gave rise to polyamide nucleic
acids (PnA). PnA are biologically stable and hybridize
effectively, although they do not activate rnase H. Also,
PnA are neutral molecules, which poses difficulties for
solubilization and cellular uptake [20, 21]. Aside from PnA,
third-generation asO n are n3’- n5’–phosphoramidates
(nP), in which the 2’-deoxyribose 3’-hydroxyl group is
substituted for a 3’-aminogrop [22], and morpholino-oligo-
nucleotides (MF), whose backbone is based on morpholine
and a dimethylamidephosphate linker [23]. On the molecu-
Fig. 1. How
antisense oligo-
nucleotides (asON)
work. (a) RNA is
cleaved as a part of
a heteroduplex with
the asON by RNase
Н. (b) Blockage of
translation caused
by binding of the
oligonucleotide onto
the mRNA

46 | ActA nAturAe | № 2 2009
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Dicer
long dsRNA
siRNA
RISC
ATP
ADP + Pi
mRNA – target
Argonaute 2
mRNA –
destruction
lar level, these oligonucleotides block translation by way
of the asOn binding to the target mrn A and/or by modu-
lating splicing. [23]. nP and MF oligonucleotides are used
primarily for developmental biology studies on zebra fish
(Danio rerio) embryos [24].
Some of the more promising chemically modified oligo-
nucleotides are LnAs (Locked nucleic Acids). these are
oligonucleotides with an additional structural element, a
2’-О,4’- c-methylene linker, which fixes the sugar residue
in the С3’-endo-conformation [25,26]. LnA are resistant
to degradation by nucleases and have a very high affinity
towards nucleic acids. the promise of LnA use in vivo is
supported by their extremely low toxicity when injected
intravenously or microinjected directly into the brains of
animals. [27].
rnA interference ( rnAi) was first discovered on the
nematode Caenorhabditis elegans ( C. elegans) as a biologi-
cal response to the introduction of foreign double-stranded
rnA (dsrnA). this response brought on the specific sup-
pression of the respective genes’ expression (gene silenc-
ing) [28]. rnAi is an evolutionarily conserved mechanism
which allows the organism to defend itself against an inva-
sion of foreign rnA such as viruses [29, 30]. After entering
the cell, exogenous dsrnA is processed into small interfer-
ing rnA (si rnA) by an intracellular ribonuclease called
Dicer [31]. these si rnAs are about 21–22 nucleotides long
and are incorporated into the multiprotein rISc complex
(rnA-induced silencing complex). si rnA that are part of
the rISc complex specifically bind to the complementary
mrnA sequence, which is then degraded by the ribonu-
clease Argonaute 2, which is also a member of the rISc
complex (Fig. 2) [30, 32]. After that, the sirnA molecules
are used repeatedly to destroy more molecules of the com-
plementary mrnA, which leads to very efficient gene si-
lencing [32]. the specific abrogation of gene expression can
be achieved by using synthetic sirnA, or sirnA enzymati-
cally constructed in vitro, as well as by using short hairpin
rnA (sh rnA), which are expressed in the cell from D nA
templates obtained by Pcr or included into DnA vectors
[33].
During the 1980s, catalytic rnA molecules were dis-
covered. these molecules could cleave rnA and are called
ribozymes [34]. naturally occurring catalytic rnA are di-
vided into large and small ribozymes. Large ribozymes in-
clude rnA which is encoded by introns of groups I and II
and also the rnA subunits of rnase P. Small ribozymes are
hammer-head-like ribozymes, hairpin ribozymes, hepatitis
D virus ribozymes, and Varkud Satellite rnA ribozymes
[35, 36]. rnA degradation by ribozymes is a 3-step process.
First the ribozyme binds to a complementary sequence by
forming classical Watson- crick base pairs; then it cleaves
the rnA substrate at a specific site; and, finally, it releases
the degradation products (Fig. 3a) [36].
Almost all the ribozyme types are being tested as thera-
peutic drugs, but the hammer-head-like ribozymes are be-
ing used more, because they have been studied more exten-
sively [35]. this ribozyme cleaves the target rnA primarily
at the nuH triplet ( n is any nucleotide and H is any nu -
cleotide except guanosine), with A uc and G uc sequences
being the most effective processing sites [37]. Another ri - Fig. 2. How RNA interference happens

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№ 2 2009 | ActA nAturAe | 47
a ribozyme action
mRNA – target
active ribozyme active DNAzyme
mRNA – target
DNAzyme actionb
mRNA –
destruction
mRNA –
destruction
bozyme often used in therapeutic studies is the hairpin ri-
bozyme [38]. the hairpin ribozyme cleaves the target rnA
at the n*Guc sequence (n is any nucleotide).
DnA molecules which exhibit catalytic activity have yet
to be discovered in nature. In 1997 Santoro and Joyce, us -
ing the SeLeX in vitro selection procedure, obtained oli-
godeoxyribnucleotides that could catalyze rnA cleavage.
these molecules were named deoxyribozymes or «10-23»
DnAzymes [39]. «10-23» D nAzymes are single-stranded
DnA molecules which have a conserved catalytic core of
15 nucleotides flanked by two variable oligonucleotide se-
quences. these flanking sequences facilitate the formation
of a complementary complex with the target rnA. (Fig. 3b)
[39]. rnA molecules are cleaved between the unpaired pu-
rine and the paired pyrimidine in the presence of magne-
sium ions. the most effective cleavage is achieved at the A u
and Gu sites.
TARgET gENES FOR DRugS BASED ON
gENE-TARgETED NuCLEIC ACIDS
A key role in carcinogenesis is played by the change in
the expression levels of certain genes whose anomalous
expression leads to the defective regulation of cell prolif-
eration, apoptosis, differentiation, and invasion. [40]. At the
molecular level, malignant transformation is a complicated
cascade of reactions; therefore, the effects of oncogenes are
often multifunctional and tightly interconnected [40]. Since
oncogenes are transcription factors and components of the
signal transduction machinery of the cell, they are involved
in many regulatory pathways, such as cell proliferation, the
inhibition of apoptosis, invasion, etc. the main target genes
for gene-targeted therapy are listed in table 1.
Firstly, malignant cell growth is based on the autonomous
and unlimited proliferation of the cell clone. that is why re-
searchers are most interested in genes which control prolif-
eration and cell cycle progression such as c-myc, ras, genes
encoding PKc-α (protein kinase С-α ) and IGF-1r (insu-
lin growth factor-1 receptor). Another effective approach
would be to target the programmed cell death system by
inhibiting the expression of antiapoptotic genes such as bcl-
2, survivin, etc.
Proteins of the ras family (K-ras, H-ras, and n-ras) are
among the best studied molecules involved in transducing
signals from tyrosine kinase receptors to the nucleus [41].
Overexpression or a point mutation of the ras gene found
in certain types of oncological diseases causes the ras pro-
tein to lose its ability to dephosphorylate; therefore, it stays
constantly activated, imitating and transducing signals that
stimulate proliferation and promote tumor-cell survival [42].
Genes of the ras family are good targets for gene-targeted
inhibition therapy. notably, ribozymes have a characteristic
way of inhibiting the activity of the ras oncogene which in -
volves an increase in the degree of tumor cells’ differentiation
[43–47]. But the leader among the gene-targeted drugs which
suppress ras oncogene expression is asOn ISIS 2503. the re-
sults of phase II clinical trials for the combined use of asO n
ISIS 2503 and gemcitabine were announced in 2004 [48].
the important role of the c-myc oncogene in cell prolif-
eration and malignant transformation was discovered in the
late 1970s by Bishop [49], and this protein was among the
first that were tested as a target for antisense therapy. the
premier drug which regulates c-myc expression is a mor -
pholine oligonucleotide asO n AVI-4126, which blocks the
production of the protein by steric inhibition of translation,
as opposed to rnase H activation [50]. this drug has suc-
cessfully passed preclinical trials and has been shown to be
well tolerated by patients. It is now in the second phase of
clinical trials [51, 52].
the protein kinase c gene family is a group of serine-
threonine kinases which are involved in regulating vital cel-
Fig. 3. Processes of
ribozyme action (a) and
DNAzyme action (b)

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Table 1. A list of target genes for drugs based on gene-targeted nucleic acids
Carcinogenic
events Target gene Function Drugs used to suppress
function
Proliferation
Ras oncogenes
(K-ras, H-ras и N-ras)
Part of the cellular signal transduction system and regulates a
wide range of processes, such as proliferation, differentiation, and
survival [42]
asOn, ribozymes, sirnA
c-myc Activates the proliferation of tumor cells (regulates the cell cycle
and telomerase activity) [49, 160] asOn, sirnA, DnAzymes
PKc-α Is involved in the cellular signal transduction and controls prolifera-
tion and cell survival [53, 54]
asOn, ribozymes,
DnAzymes
clusterin
Is involved in lipid transport, cell division, and apoptosis; supports
cell survival in response to therapy; and increases tumor drug
resistance [57, 58]
asOn
IGF-1r Activates the MAPK and PI-3K signal pathways, which stimulate
proliferation and mitogenesis and inhibit apoptosis [61–63] asOn
Blocking of
apoptosis
bcl-2 negativly regulates apoptosis by blocking the excretion of cyto-
chrome c from the mitochondria into the cytoplasm [67] asOn, sirnA, ribozymes
Survivin
regulates cell division (interacts with microtubules in the mitotic
spindle and promotes mitotic entry through G2/М checkpoint)
and suppresses apoptosis (inhibits the inner caspase-9-dependant
apoptosis pathway) [73, 74]
asOn, ribozymes
bcr-abl Stimulates a mitogenic and antiapoptotic signal mediated by ras-
regulated signal pathways [81, 82]
ribozymes, DnAzymes,
sirnA
с-raf Activates the MAPK/erK cascade and negative regulation of
apoptosis by inactivating the proapoptotic Bad protein [88, 90] asOn
Drug resistance
MDr1
Forms transmemrane channels for AtP-dependant expulsion of
drugs out of the cell, which endows tumors with drug resistance
[95–97]
asOn, sirnA, ribozymes
γ – glutamylcysteine
syntetase (glutathione
system)
Intracellular detoxication of anticancer drugs [95] ribozymes
Dysfunction of
tumor suppressor
genes
DnMt1 Hypermethylation and inactivation of tumor suppressor genes
[102, 103] asOn
Increase of tumor
cell lifespan htert
Hyperactivation of the telomere repeat elongation machinery
and, as a result, an increase in the malignancy and the lifespan of
transformed cells [109]
asOn, ribozymes
DnA synthesis
arrest RRR2 or RRM2
controls the amount of deoxyribonucleotides needed for DnA syn-
thesis via regulating the conversion of ribonucleotides into deoxy
ribonucleotides [113]
asOn, sirnA
tumor
angiogenesis
Flt-1 (VeGFr) Active neovascularization and suppression of the anti-tumor
immune response [119, 120]
asOn, ribozymes,
DnAzymes, sirnA
neu
(Her2 or erbB2, eGFr
family)
Activation of signal transduction pathways, which lead to the
stimulation of tumor progression events such as proliferation, inva-
sion, and apoptosis inhibition [127,128]
asOn, ribozymes
eIF4e Increases the translation of growth factors such as VeGF, С-myc,
surviving, etc. [133 - 135] asOn
Ptn Is a growth factor, promotes active tumor growth and vasculariza-
tion [137 - 139] ribozymes
ALK Is a tyrosine kinase Ptn receptor, facilitates its function, thus
promoting tumor vascularization [141] ribozymes
tumor invasion
MMP9 elimination of components of the extracellular matrix and basal
membrane and promotes tumor invasion [144 – 146] ribozymes, sirnA
FGF-BP Activation of FGF-2, which induces tumor cell proliferation and
increases the invasive and angiogenic potential [148] ribozymes
egr-1 Activates proliferation and tumor invasion and is involved in estab-
lishment of the MDr phenotype [154,155] DnAzymes
FAK regulates adhesion and invasion into the extracellular matrix [158] sirnA
cXcr4 Stimulates metastatic processes [159] sirnA

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№ 2 2009 | ActA nAturAe | 49
lular functions such as differentiation, cell-cell interactions,
secretion, cytoskeleton functions, gene transcription, prolif-
eration, and apoptosis [53]. Among a dozen of isoforms of this
protein, only PKc-α is shown to be involved in cell survival,
proliferation and apoptosis [54]; it is also actively involved in
neoplastic transformation of the cell. the best candidate for
selective suppression of the tumor-specific PKc-α isoform
turned out to be the thiophosphate asOn ISIS 3521 (Affini-
taktM, uSA), which selectively binds the PKC-α mrnA and
does not interact with the other, non-oncogenic members of
the PKc family [55]. this drug has been approved for clini-
cal trials.
clusterin was first described in 1983 as a secretory glyco-
protein [56] associated with a wide variety of physiological
and pathological processes, such as lipid transport, tissue
transformation, cell membrane defense, apoptosis, and the
complement system function [57]. Later research showed
that clusterin is a chaperone-like protein which increases
cell survival in response to stress [58]. the inactivation of
this gene by gene-directed drugs could compromise the
DnA repair-system response to external hazards, such as
chemotherapy. this turned out to be true; for instance,
asOn OXG-011 increases the toxic effect of paclitaxel on
tumor cells in mice [59] and is on the clinical path in combi-
nation with chemotherapeutic drugs [60].
IGF-1r (insulin growth factor-1 receptor) is a trans-
membrane protein kinase which promotes independent cell
growth [61]. Anomaly in IGF-1R expression is well known to
be connected with carcinogenesis [61]. It was demonstrated
that IGF-1R overexpression promotes the development of
P-glycoprotein- and Bcl-2-mediated multiple drug resist-
ance (MD r) [61,63]. Suppressing the IGF-1R gene with a
gene-targeted antisense oligonucleotide prevents tumor for-
mation in mice in ex vivo1 experiments [64], which confirms
the idea that asO n are promising drugs to counteract the
cancer cell defense system.
the Bcl-2 protein is the major representative of a fam-
ily of pro- (Bax, Bak, Bad) and anti- (Bcl-xl, Mcl-1) apop-
totic factors and was first discovered in B-cell lymphoma
cells in 1985 [65]. Bcl-2 gene expression is associated with
aggressive tumor behavior in response to chemo- and radio -
therapy [66]. excess Bcl-2 protein promotes the inhibition of
mitochondrial membrane depolymerization, thus blocking
apoptotic mechanisms which are triggered by the therapy
[67]. Suppressing bcl-2 expression with gene-targeted oli-
gonucleotides facilitated apoptosis induction in tumor cells
and increased cell sensitivity to apoptosis-inducing chemo-
therapeutic drugs [68, 69]. AsOn G3139 showed a consider-
able therapeutic effect and is currently in phase III clinical
trials [70–72].
Survivin is a member of the apoptosis-inhibiting protein
family (IAP). Despite the fact that its precise function in the
cell is yet unclear, it was shown that this protein is involved
in regulating cell division and apoptosis [73]. Survivin se-
lectively inhibits the inner caspase-9-dependant apoptotic
pathway [74], and it also bypasses apoptosis mechanisms
1 Here and further in the text, an ex vivo experiment is defined as tumor
material being removed from the organism, treated by a gene-targeting
drug in vitro, and introduced back into the organism.
by interacting with the microtubules of the mitotic spindle,
which promotes mitotic entry through the G 2/M checkpoint
[75] and in turn stimulates anomalous cell cycle progres-
sion. Suppressing the survivin encoding gene with asO n led
straight to apoptosis induction and tumor cell death [76, 77].
the inhibitory effect of a ribozyme on the activity of the
gene in the absence of additional apoptosis inducers did not
affect the viability of cells [78–80]. there have been no in
vivo studies of survivin as a target gene so far.
the chromosome translocation t(9;22) combines two
independent genes – bcr, which is located on the human
chromosome 22, and abl, located on chromosome 9 – and
thus forms a hybrid oncogene [81]. Like the original abl
gene, the chimeric bcr-abl gene shows increased kinase ac-
tivity. By phosphorylating certain cell factors, Bcr-ABL
facilitates malignant transformation and blocks apopto-
sis [82]. Specifically, the appearance of bcr-abl can lead to
myeloid or lymphoid leukemia [83]. the hybrid nature of
this protein presented several difficulties in trying to af-
fect its production. In some cases, the gene-targeting drugs
suppressed not only the chimeric gene expression, but also
that of the original abl [84]. It turned out that Maxizyme,
a double ribozyme which could cleave the target mrnA
at two sites, was an effective tool for circumventing this
non-specificity [85]. the drug Imatinib, which specifical-
ly inhibits tyrosine kinase, was developed for preventing
chronic myeloleukemia and can be used to effectively con-
trol leukemia progression [86]. However, there are known
cases of the disease where the tumor is resistant to Imat -
inib because of point mutations in the gene or for other
reasons. In such cases, rnAi methods can be helpful. ex-
periments using sirnA have shown that Bcr-ABL+ cells
can be sensitized to Imanitib by rnAi [87].
Proteins of the raf family are serine-threonine kinases
which transduce signals from a wide variety of membrane
growth factor receptors to apoptosis regulators. It was de-
termined that the functionally active raf-1 kinase acti-
vates the MAPK/erK pathway (mitogen-activated pro-
tein kinases/extracellular signal-regulated kinases) [88],
takes part in the signaling pathways of proliferation and
cell survival via nF-κB [89], and inhibits the proapoptotic
Bad protein [90]. thus, raf-1 is at the center of a network
of vital signalling pathways; thus, mutations or defective
expression of the raf-1 gene play a considerable carcino -
genic role in cell transformation. In addition, raf-1 is an
effector of the protein product of the ras oncogene [91],
which is often found to be mutated in malignantly trans-
formed cells; this is why therapy directed at suppressing
raf-1 can prove to be effective against ras-mediated neo-
plasias. the most promising results of specific c-raf gene
suppression and the corresponding antitumor effect were
obtained using asOn. Investigators reported the results of
phase II clinical trials in 2002 using a drug based on a thio-
phosphate asOn [92-94].
In general, about 30% of the patients who receive tradi -
tional chemotherapy develop multi-drug resistance related
to the MDR1 gene [95–97], which encodes the P-glycopro -
tein. P-glycoprotein is a member of the AB c-transporter
protein superfamily, which uses A tP-hydrolysis to expel
chemotherapeutic drugs from the cell. P-glycoprotein hy-

50 | ActA nAturAe | № 2 2009
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peractivity endows tumor cells with resistance to a wide
variety of chemotherapeutic drugs. Because of this, inacti-
vating the MDR1 gene can facilitate chemotherapeutic drug
retention in cells and cause their death. MDR1 is a fairly
common target for gene-targeted oligonucleotide drugs.
Among these, ribozymes and sirnA are currently consid-
ered the most effective. these drugs almost entirely blocked
tumor growth in mice [98, 99].
One of the most important cytostatic drug neutralizing
systems of the cell is the glutathione system. Glutathione
is a nonprotein thiol which has a sulfhydryl group that can
interact with the reactive group of a drug and, as a result,
form a conjugate of glutamine with the drug [95]. these
conjugates are less reactive, more soluble, and are expelled
from the cell by transporter proteins [95]. thus, the acti-
vation of the glutathione system genes can cause tumor
drug resistance [100]. to circumvent MDr caused by the
glutathione system, a ribozyme that effectively restored
tumor cell sensitivity to chemotherapeutic drugs was con-
structed [101].
Many studies have shown anomalous methylation at cer-
tain sites in the genome in tumor cells [102]. the enzyme
DnMt1 (DnA methyltransferase 1) catalyses the transfer
of a methyl residue from S-adenosinemethionine to the 5th
position of the cytosine residues in cpG islets, affecting the
expression of certain genes [103]. It was shown both that
tumor cells have elevated DnA methyltransferase activ-
ity [104] and that the initiated hyperactivity of this enzyme
leads to malignant transformation [105]. Furthermore, it is
alleged that anomalies in methylation processes are a key
factor in determining the tumors’ response to chemotherapy
[106]. Antisense inhibition of DNMT1 gene expression led to
the restoration of the function of tumor suppressor genes
and increased tumor cell death [107]. the artificial antisense
oligonucleotide MG98 is currently in phase II clinical trials
[108].
the telomerase reverse transcriptase restores the tel-
omere length by adding tandem repeats (ttAGGG) and is
needed to fully replicate the ends of chromosomes [109]. It
was demonstrated that the hyperactivation of hTERT (hu-
man telomerase reverse transcriptase) and carcinogenesis
are highly correlated [110]. In order to suppress the gene’s
activity, several differently modified asOn were construct-
ed, including a 2’-O-methyl asOn, PnA [111] and a 2’5’-oli-
goadenilate oligonucleotide [112]. the 2’-O-methyl asO n
suppressed hTERT expression in a cell culture by 97% [111],
and the 2’5’-oligoadenilate oligonucleotide caused a 50% re-
gression of tumors [112].
ribonucleotidereductase catalyzes the synthesis of
2’-deoxyribonucleotides from the corresponding ribonu-
cleoside 5’-diphosphates. this is the limiting step in the
formation of 2’-deoxyribonucleoside-5’-triphosphates,
precursors in DnA synthesis [113]. the r2 subunit of ri-
bonucleotidereductase ( rrr2) is synthesized in the late
G1 phase and the early S phase and is a key factor in de-
termining the rate of D nA replication [113]. Also, it is well
known that rrr2 plays a significant role in determining
the malignant potential of cells by acting synergistically
with certain oncogenes and being connected with the mem-
brane protein raf-1 and the mitogen-associated protein
kinase 2 (MAPK2) [114, 115]. For this reason, the specific
suppression of the RRR2 mrnA can cause a whole array
of antineoplastic effects via a wide variety of mechanisms.
AsOn Gt I-2040, which suppressed RRR2 expression,
caused a 98% regression of renal carcinoma transplanted
into mice [116]. clinical trials showed that this drug was
well-tolerated by patients [117].
In the early 70s Folkman proposed that the growth of
solid tumors and metastasis are critically dependant on
angiogenesis – the formation of new blood vessels from
the surrounding vascular network [118]. the pathological
growth of new vessels promotes solid tumor progression
and metastasis. Over the last few decades, the main media-
tors of angiogenesis have been identified and character -
ized, providing researchers with novel targets for cancer
therapy. Among the numerous neoangiogenic factors, the
most prominent is V eGF (vascular endothelial growth fac -
tor) [119]. By activating receptors V eGFr-1 and VeGFr-2,
VeGF induces the activity of intracellular signaling tyrosine
kinases, which play a central role in stimulating the prolif-
eration of endothelium cells [120]. At least 5 V eGF isoforms
are generated by alternative splicing: VeGF 206, VeGF 189,
VeGF165, VeGF145, VeGF121 [121, 122]. It has been shown that
increased angiogenesis and tumor progression are associated
with the overexpression of isoform V eGF165 [123]. Specific
oligonucleotides and sirnA were developed in order to sup-
press the expression of the V eGF-encoding gene [124, 125].
An interesting approach towards inhibiting V eGF activity
was the construction of ribozymes, and later D nAzymes,
targeted at inhibiting the expression of Flt-1, which encodes
the VeGF receptor VeGFr-1, and the KDR gene, encoding
VeGFr-2 [126]. Among all the proposed strategies of V eGF
inhibition, this approach proved to be the most effective,
and the ribozyme-based drug Angiozyme, targeting the Flt-
1 gene, is currently in phase II clinical trial [35].
the protooncogene neu, also known as HER-2/erbB-2 or
NGL, encodes a transmembrane receptor that exhibits ty-
rosine kinase activity, which is important for intracellular
signal transduction [127]. normally, the H er-2 protein is
not produced in most human tissues, and in neoplasias this
receptor exhibits tyrosine kinase activity even when there
is no ligand to activate it [128]. the overexpression of Her-
2 in carcinogenesis is often attributed to the amplification
of the corresponding gene [129]. the recently developed
drugs Herceptin and rituxanar are based on monoclonal
antibodies that bind the Her-2 receptor, but clinical trials
showed some negative side-effects, the worst being cardio-
toxicity [130, 131]. this is why the gene-targeted therapy
of Her-2-mediated neoplasias looks so promising. Of all the
drugs based on nucleic acids and used to suppress H er-2
activity, the drug Herzyme, based on a hammer head ri -
bozyme, is currently in clinical trials. Phase I clinical trials
showed that the drug is well tolerated by patients [132].
the eukaryotic translation initiation factor 4 e (eIF4 e)
binds to the capped 5’ terminus of cellular mrnAs and de-
livers them into the eIF4 e translation initiation complex.
this complex reads the mrnA sequence in the 5’-3’ direc-
tion and unwinds the secondary m rnA structure in the
5’-untranslated region, thus uncovering the translation start
codon and promoting ribosome binding [133, 134]. In normal

reVIeWS
№ 2 2009 | ActA nAturAe | 51
conditions, eIF4 e activity is down-regulated by a specific
eIF4e- binding protein 4e-BP (4e-binding protein). Malig-
nant transformation is often accompanied by eIF4e overex-
pression or by the phosphorylation of 4 e-BP. this leads to
the release of an active eIF4e which forms the eIF4e trans-
lation complex [135]. notably, many researchers have shown
that hyperactive eIF4 e in tumors mostly enhances the
translation of proteins which are involved in tumor progres-
sion such as Bcl-2, survivin, cyclin D1, c-myc, and V eGF
[133, 134]. Because of this, the specific suppression of eIF4 e
activity can turn out to be very useful in inhibiting tumor
progression. An asOn complementary to eIF4e showed good
results. When injected into tumor-bearing mice, it caused a
10-fold regression of the tumor and had no noticeable side
effects [136].
Pleiotropin (Ptn) is a secreted growth factor which is pro-
duced in large quantities during the development of the nerv-
ous system and “turned off” in adults [137], except in some
cancer patients [138]. Ptn is an active mitogen for fibroblasts
and epithelial cells [137, 138]. Also, it can induce the release
of active proteolytic enzymes from endothelial cells [139].
these data point to the potentially crucial role of Ptn in an-
giogenesis. In order to suppress the expression of PTN in tu-
mor cells, a ribozyme complementary to the PTN mrnA was
constructed. It showed considerable antitumor and antimet-
astatic activity [140]. Moreover, it was recently discovered
that the inhibition of the expression of anaplastic lymphoma
kinase (APK), which causes the development of anaplastic
lymphoma [141] and functions as a pleiotropin receptor [142],
did not only cause tumor regression, but also caused a two-
fold increase in mean survival time in mice [143].
tumor progression is accompanied by the tumor’s abil-
ity to spread beyond the boundary of its own tissue and to
continue to grow into nonrelated tissues. Modulating the
expression of genes which are involved in stimulating mi -
gration and invasion by means of gene-targeted drugs is a
strategy that is often used by researchers. Matrix metal-
loproteinase-9 (MMP9) can initiate the degradation of cer-
tain components of the extracellular matrix and the basal
membrane (collagens IV and V, elastin, entactin, casein, and
galectin) [144, 145], which promotes the epithelial-mesen-
chymal transition of tumor cells and stimulates metastasis
[146]. the suppression of metalloproteinases by ribozymes
partially abrogates metastasis and increases the mean sur-
vival time of tumor-bearing mice, but it does not cause tu-
mor regression [147].
the fibroblast growth factor (FGF) is interesting to re-
searchers because it is a powerful mitogen which induces
differentiation and angiogenesis during development and
also stimulates tumor cell invasion [148]. normally, adults
produce only a small quantity of FGF, but during certain
oncological diseases, FGF production is elevated [149]. Se-
creted FGF binds tightly to heparan sulphate proteoglycans
in the extracellular matrix, thus blocking FGF activity [150].
One of the mechanisms of FGF release is binding to FGF-
BP (FGF-binding protein), which mobilizes and activates
FGF [150]. Studies have shown that FGF-BP is expressed in
certain carcinomas [151] and also promotes the conversion
of nononcogenic FGF-expressing cell line SW-13 to onco-
genic and angiogenic phynotype [150]. A ribozyme designed
to suppress the FGF-BP gene expression effectively abro-
gated tumors in mice [152].
the early growth response factor-1 (eGr-1) is a typi-
cal representative of a family of transcription factors which
possess a “zinc-finger” structural domain [153]. eGr-1 ac -
tivity is induced by a number of external and intracellular
stimuli such as growth factors, cytokines, ultraviolet light,
ionizing radiation, etc [153]. It has been shown that eGr-1
is involved in multiple regulatory pathways in tumor cells.
Its activity is involved in the development of malignant
transformation [154], in the regulation of MDR1 gene tran-
scription [155], and in the negative response to estrogen in
mammary gland carcinoma [156]. In order to suppress eGr-
1 gene expression, a D nAzyme, which caused a 3-fold de -
crease in tumor size in mice, was designed [157].
the focal adhesion kinase (FAK) is a non-receptor tyro-
sine kinase, located in integrin clusters, through which the
cytoskeleton interacts with proteins of the extracellular ma-
trix (focal adhesion sites). FAK receives signals from growth
factors and adhesion factors and transmits them into the
cell. FAK is an important mediator of signal transduction
pathways, regulating proliferation, migration and cell vi-
ability and it is often overexpressed in tumor cells. si rnA
effectively suppresses FAK activity and tumor growth in
mice [158].
In mammary gland tumors, the transformed cells express
the chemokine receptor cXc (cXcr4), which causes me-
tastasis into organs containing a lot of cXcr4 ligands. the
inhibition of cXcr4 expression with si rnA suppresses the
adhesive and invasive properties of tumor cells [159].
APPLICATION gENE-TARgETED NuCLEIC ACID
DRugS IN TISSuE CuLTuRES, ExPERIMENTAL
ANIMAL MODELS AND CLINICS
Drugs based on nucleic acids have attracted the attention
of researchers for a long time, being potentially useful for
gene-targeted cancer therapy due to their ability to interact
with the key pathways of carcinogenesis. table 2 sums up the
main results of in vitro studies in this area and table 3 com-
bines the data of preclinic in vivo tests and clinical trials. In
this section, we describe the development of drugs based on
gene-targeted oligonucleotides which specifically inhibit the
functions of target genes involved in carcinogenesis.
Ras
In vitro. the established location of point mutations in the
ras oncogene mrnA allows the development of oligonu-
cleotides specifically targeted at the mutated sites in the
mrnA target. Such drugs efficiently switch off oncogene
expression. the first drug developed for ras oncogene sup-
pression was a thiophosphate olignucleotide asO n (now
known as ISIS 2503) targeted at the initiation codon of H-
ras mrnA [161]. ISIS 2503 treatment of cervical carcinoma
HeLa cells transfected with a plasmid encoding a gene for
ras-luciferase fusion protein led to 98% suppression of the
reporter gene. In a study by chinese researchers, treatment
of human hepatoma cells by a thiophosphate asOn for 5
days caused an 87.8% inhibition of tumor cell growth [162].
Additionally, a blockage of H-ras-dependant entry of tumor
cells into the S-phase of the cell cycle was observed, and

52 | ActA nAturAe | № 2 2009
reVIeWS
DnA fragmentation detected in the treated cells indicated
the initiation of apoptosis [162].
the antitumor potential of ribozymes targeted at ras
family genes is being actively investigated. In the melano-
ma, throat carcinoma and bladder tumor cell models, several
research groups have demonstrated that H-ras ribozymes
induced apoptosis, inhibited the proliferation of tumor cells,
and helped reestablish cell differentiation [44–47].
An alternative approach to the suppression of ras-family
gene expression uses rnAi technology. retrovirus-mediated
expression of sirnA targeted at H-ras and K-ras mrnA ef-
fectively suppressed the synthesis of these proteins in ovar-
ian and pancreatic carcinoma cells [163, 164]. the tumor pro-
liferation activity decreased as the cells accumulated in the
G0/G1 phase of the cell cycle, their proportion reaching 66.2%
[163] and the number of apoptotic cells increasing from 4% to
21% [163]. Zhang and coauthors used an adenoviral system of
K-ras-sirnA delivery into cells and obtained an 80% decrease
in the amount of K-ras protein in lung tumor cells, along with
the suppression of tumor-cell proliferation [165].
In vivo. the transfection of hepatocellular carcinoma cells
by H-ras asOn produced a decrease in the average tumor
weight in mice [162, 166] and inhibited metastasis [166]. the
antitumor efficiency of asOn ISIS 2503 in mice with pros-
tate tumors increased upon the addition of L nA nucleotides
into the oligonucleotide sequence [167]. AsO n ISIS 2503 was
approved for clinical trials. In a phase I trial, the patients
received 10 mg per kg asO n ISIS 2503 injections for 14 days
[168]. After a one week recess, the injection course was re-
peated. AsOn ISIS 2503 did not display any marked toxicity
and prevented the further progression of the disease. the
results of phase II trials, performed on patients suffering
from pancreatic adenocarcinoma have established that com-
bined treatment by asOn ISIS 2503 and Gemcytabine is well
tolerated and demonstrated a positive response to treatment
in 10.4% of patients [48].
the use of anti-H-ras ribozymes in vivo led to a signifi-
cant retardation of tumor growth in mice and to a decrease
in their invasive potential, as well as a two-fold increase in
mouse lifespan [43–45]. Kijima and coauthors developed a
ribozyme targeted at codon 12 of the mutant K-ras gene
(GGt triplet substituted for Gtt) and obtained a recom-
binant adenovirus expressing this ribozyme [169]. An intra-
tumor injection of this drug into athymic mice with a trans-
planted pancreatic carcinoma caused tumor regression in
68% of mice [169].
In vitro treatment of tumor cells with a retroviral vector
containing sirnA targeted at K-ras и H-ras mrnA caused
a complete inhibition of pancreatic carcinoma growth and
80% inhibition of ovary tumors in mice [163, 164]. A one-
time intratumor injection of adenoviral sirnA targeted at
K-ras caused a 45% inhibition of lung tumor development,
and multiple injections completely stopped tumor growth in
8 out of 10 mice. In these experiments, the apoptotic activity
of tumor cells showed a 2.8-fold increase [165].
C-myc
In vitro. the first drug developed for specific suppression
of the c-myc protooncogene was a phosphodiester, asO n,
which caused a 50% decrease in the protein level in leuke-
mia cells and inhibited their proliferation by 50% after 5-day
incubation with the olignucleotide [170]. Watson et al. de-
veloped a thiophosphate asO n which demonstrated a more
prolonged (up to 9 days) and effective (75%) inhibition of
breast cancer cells proliferation and caused a 95% inhibition
of estrogene-induced overexpression of c-myc gene [171].
the next step involved the substitution of the thiophos-
phate backbone for a morpholino-phosphorodiamidate one.
Such an anti-c-myc-asO n not only caused a decrease in the
protein level, but also a complete G 0/G1 cell cycle blockage
[172].
A “hammer head” type ribozyme was developed in order
to suppress c-myc activity. transfection by a retroviral vec-
tor encoding the ribozyme led to a 1.7-fold decrease of the
protein level in hepatoma cells and a 1.85-fold decrease in
the proliferation potential of the cell [173].
the ability to suppress c-myc protein hyperactivity was
evaluated for gene-targeted sirnA drugs. It was shown that
sirnA caused a 60–92% decrease in the m rnA level and
a 55–83% decrease in c-myc protein synthesis [174, 175].
the partial abrogation of c-myc expression was associated
with a 2.5-fold suppression of cell growth in human epi-
dermoid carcinoma KB-3-1 and with a complete prolifera-
tion blockage in SK-n-Mc neuroblastoma [175]. In order to
improve the stability and facilitate intracellular delivery,
a special poly-DnP-rnA (poly-2-О-(2,4-dinitrophenyl)-
oligoribonucleotide) was designed. this drug lowered the
c-myc mrnA level to 15% in mammary and lung adenocar-
cinoma tumor cells [176].
In vivo. In vivo experiments showed that AVI-4126 (AVIBi-
oPharma, united States), a morpholino-oligonucleotide de-
veloped for inhibiting c-myc expression, causes an 80% de -
crease in prostate tumor growth in athymic mice [50]. this
drug is currently in phase II clinical trials [52]. Phase I trials
showed that AVI-4126 had no serious side effects for healthy
people who received a single intravenous injection of AVI-
4126 with a dosage of 90 mg [51]. Also, AVI-4126 was assessed
for accumulation in the prostate and mammary tumor tissues
of patients who were injected with 90 mg of the drug (the test
was performed 24 hours after the injection using surgically
removed tumors) [51].
the inhibition of с-myc expression by rnAi looks promis-
ing in preclinical trials. Breast cancer cells transfected with a
plasmid encoding anti-c-myc-siРНК did not give rise to tu-
mors when transplanted into mice [177]. In transgenic mice
with developing lymphoma, real-time rt-Pcr detected a
15%–20% decrease in c-myc m rnA in the plasma of mice
treated by poly-D nP-rnA [176].
PkC-α
In vitro. In order to inhibit the expression of the PKC-α
gene, Dean et al. performed a thorough study of the effi-
ciency of 20 thiophosphate asOn and their 2’-О-methyl ana-
logs [55]. the most effective oligonucleotide (known under
the commercial name ISIS 3521) caused a 90–95% decrease
in the level of PKC-α mrnA, but its 2’-О-methyl derivative
did not affect the expression of the PKC-α gene, which indi-
cates that rnAse H activity is required [55].
In the glioblastoma model, the inhibitory potential of
an anti-PK c-α ribozyme was evaluated. A 73% decrease

reVIeWS
№ 2 2009 | ActA nAturAe | 53
Table 2 . Results of nucleic acid-based drugs testing in vitro
Target-genes Drug Tumor type Effect
H-ras/K-ras
asOn cervical carcinoma [161],
hepatoma [162]
A decrease in H-ras-luciferase-mrnA level by 98% [161]; inhibition of cellular growth by
87.8%, block of the entry into the S-pahse of the cell cycle, apoptosis induction [162]
ribozyme Melanoma, throat carcinoma, bladder
cancer
Decrease in H-ras expression; retardation of proliferation and an increase in the level of
differentiation of tumor cells [ 43–47]
sirnA Ovarian, pancreatic [163], lung [165]
carcinoma
80% decrease in the protein level [165], suppression of proliferation [163,164], changes in the
cell-cycle schedule, and increased number of apoptotic cells [163]
с-myc
asOn Leukemia [170], breast cancer [171] 50%–95% decrease in c-myc expression [170, 171]; complete cell cycle arrest in the G0/G1
phase [172]
ribozyme Hepatoma 1.7-fold decrease of the protein level and 1.85-fold drop in proliferative activity [173]
sirnA
epidermoid carcinoma,
neuroblastoma [175], breast cancer
and lung adenocarcinoma [176]
60–92% decrease in mrnA level and 55–85% inhibition of protein synthesis [175,176]; slow-
ing and blockage of cell division [175]
PKc-α
asOn Lung carcinoma 90–95% decrease in PKC-α mrnA level [55]
ribozyme Glioblastoma [178], prostate carcinoma
[179]
Decreases protein level by 73% and proliferative activity below 90% [178]; restores
sensitivity to cisplatin [179]
clusterin asOn
OGX-001 renal carcinoma Decreases clusterin mrnA level by 64% and increases cell sensitivity to paclitaxel by 80%
[59]
IGF-1r asOn Bladder cancer Decrease of mrnA level by 74% and protein level by 61.3% [207]
bcl-2
asOn G3139
(Genasense™, uSA) Lymphoma [183], leukemia [68, 69]
Decrease of bcl-2 mrnA level and Bcl-2 protein level by 60–80% and 80–95%, respective-
ly, hence increasing cell death rate by 76–90%; as a result of apoptosis induction, increases
doxorubicin senstivity [68, 69, 183]
ribozyme Lymphoma 5-fold decrease in mrnA level, 3-fold decrease in protein level, and 2-fold increase in
apoptosis rate [184]
sirnA cervical [185] and pancreatic [189]
carcinoma
Suppresses Bcl-2 protein synthesis by 90%, induces apoptosis in 50% of cells [185]; increases
proportion of apoptotic cells by 37% [189]
Survivin
asOn
Malignant lung mesothelioma, glioma,
breast cancer, lung adenocarcinoma
[76], thyroid cancer [77]
7-8- fold increase in caspase-3 activity, induction of apoptotic cell death in 42.5% of cells
[76]; decrease of the mrnA level by 75% and protein level by 73%, inhibits cell proliferation
by 53%, 11-fold increase in the proportion of apoptotic cells [77]
ribozyme Melanoma [78, 79], breast cancer [80]
Decreases mrnA and protein levels by 75% and 74% respectively, increases tumor cell
sensitivity to chemo- and radio-therapy, no effect without an additional apoptose inducing
stimulus [78 - 80]
bcr-abl
asOn chronic myeloid leukemia complete inhibition of cell growth, apoptosis induction [208]
Maxizyme chronic myeloid leukemia 95% decrease in the chimeric gene mrnA, apoptosis induction, tumor cell growth retarda-
tion [85]
sirnA chronic myeloid leukemia Suppression of Bcr-ABL-associated cell growth, 4-fold increase in tumor cell sensitivity to
imanitib [87]
DnAzyme chronic myeloid leukemia Suppresses protein production by 40-75% [208]
c-raf asOn Lung, colon, prostate[190, 191], ovary
[192, 193] cancer
100% suppression of С-raf protein, 80% inhibition of cell proliferation [190 - 192]; growth
suppression in various ovary carcinoma lines by 10% to 90% [193]
sirnA Bladder cancer Decrease of protein level by 37.5% [194]
MDr1,
mdr1a/
mdr1b
asOn colon adenocarcinoma [211], epider-
moid carcinoma [212]
complete MDr phenotype reversal, increases accumulation of doxorubicin in cells 6.4-fold,
promotes cell death [211, 212]
sirnA
Human epidermoid carcinoma [215],
ovary cancer cells” на “human pancre-
atic cancer and gastric carcinoma [217],
ovarian cancer cells, ovary cancer cells
[218], murine lymphosarcoma [219]
MDR1 mrnA level is down by 91% and the P-glycoprotein by 72%-83%, increases cell
sensitivity to vinblastin [99, 215], duanorubicin [217] and paclitaxel [218]
ribozyme Liver cancer reverses MDr phenotype, increases cell sensitivity to vincristin [214]
Glutathine ribozyme colon cancer Increases tumor cell sensitivity to chemotherapeutic drugs [101]
DnMt1 asOn MG98 Lung and bladder cancer restores p16 function, promotes accumulation of hypomethylated form of retinoblastoma
protein, inhibits proliferation [107]
htert
asOn Bladder cancer [111, 112] Decreases the protein level by 97%, increases cytostatic drug sensitivty, causes 3-fold
increase of proportion of apoptotic cells, activates caspase-3 [111, 112]
ribozyme Breast cancer reduces the length of the telomere tandem repeat region from 5.5 kbp to 3.5 kbp and
reduces cell growth rate [224]
rrr2
asOn
GtI-2040
(Genasense™, uSA)
Lung, bladder carcinoma, fibrosar-
coma results in total disappearance of r2 subunit mrnA
sirnA Pancreatic adenocarcinoma Increases tumor cell sensitivity to gemcytabin [198]
VEGF, Flt-1
(VeGFr1),
KDR
(VeGFr2)
asOn Breast and bladder cancer Decreases VeGF level by 45–83% and lowers cell survival [124]
sirnA Ovarian, cervical cancer, osteosarcoma reduces VEGF expression by 33–53% [125]
ribozyme
Angizyme, Sirna
ther., uSA
Lung and colon carcinomas,
breast cancer
causes specific cleavage of rnA-substrate and effectively decreases mrnA level in cell
culture [199]
DnAzyme Breast cancer Decreases VeGFr-2 level by 90% and cell survival by 34–65% via inducing apoptosis [200]
neu (Her-2/
erbB2)
asOn Ovarian and breast cancer Additive inhibition of tumor cell proliferation in combination with doxorubicin [205]
ribozyme
Herzyme Ovarian and breast cancer Lowers neu mrnA levels by 40–60%, thus inhibiting cell growth [131]
eIF4e asOn
LY2275796
non-Hodgkin lymphoma, lung, bladder,
colon, prostate and breast cancer
Decreases eIF4e protein level by 80%; lowers protein levels of Bcl-2, survivin, cyclin D1,
С-myc and VeGF; and induces apoptosis [136]
PTN и ALK
ribozyme
(anti-Ptn) Melanoma Decreases PTN mrnA level by 75% [140]
ribozyme
(anti-ALK) Glioblastoma Decreases Ptn activity [143]
MMP9 ribozyme Prostate cancer causes complete degradation of MMP9 mrnA [220]
sirnA Juvenile osteosarcoma 50% decrease in cell migration [221]
FGF-BP ribozyme Prostate and colon carcinoma [152, 222] 80% suppression of FGF-BP protein synthesis; slowing down of tumor cell proliferation [152, 222]
eGr-1 DnAzyme Breast cancer 6-fold decrease of protein level, blocking of proliferation, and 3-fold drop in tumor cell
invasion activity [157]
FAK sirnA Prostate and breast cancer Inhibits tumor cell adhesion, migration, and proliferation [158]
cXcr4 sirnA Breast cancer Inhibits cell migration and invasion by more than 70% [225]

54 | ActA nAturAe | № 2 2009
reVIeWS
in the protein level after ribozyme treatment was demon-
strated, and proliferative activity of the tumor cells was
down by 90% as compared to 50% after treatment with a
control ribozyme [178]. In another study, a PKc-α -targeted
ribozyme re-established prostate carcinoma cells’ sensitiv-
ity to cisplatin-induced apoptosis, increasing its rate 2.6–
3.2-fold [179].
In vivo. Intravenous injections of ISIS 3521 to mice with
three different heterotransplants (lung carcinoma, bladder,
and colon cancer) caused complete tumor growth suppres-
sion in mice at a comparatively low dosage of 0.06–0.6 mg/
kg [55]. In phase I clinical trials of asO n ISIS 3521, several
different treatment schedules were investigated. the main
cause of toxicity was thrombocytopenia and fatigue [180]. In
phase II trials with a recommended treatment schedule of
2 mg/kg/day for 3 weeks, an objective treatment response
was documented for one patient with ovarian carcinoma,
and 2 patients with ovarian carcinoma displayed tumor
regression [181]. However, the treatment of patients with
metastatic colon carcinoma did not produce any statistically
significant responses [182].
CLuSTERIN
In vitro. In the inhibition of clusterin expression, thiophos-
phate asOn affected a 64% decrease in gene expression and
an 80% increase in paclitaxel sensitivity in renal carcinoma
[59].
In vivo. 2’-methoxyethyl-modified thiophosphate asO n
OXG-011 combined with paclitaxel caused a two-fold drop
in the volume of renal carcinoma in mice [59]. Phase I of the
clinical trials of asOn OXG-011 at the dose of 640 mg re-
duced clusterin levels in human tumor tissue [60]. currently,
the therapeutic potential of asO n OXG-011 combined with
chemotherapy is being investigated in phase II clinical trials.
Bcl-2
In vitro. Anti-m rnA drugs targeted at the antiapoptotic
Bcl-2 protein emerged as undisputed leaders among the
gene-targeted oligonucleotides used for cancer treatment.
An 18-bp phosphodiester asO n, complementary to the first
6 codons of the open reading frame of bcl-2 mrnA, was de-
veloped by Kitada and coauthors. this oligonucleotide al-
most completely blocked Bcl-2 protein synthesis in lympho-
ma cells [183]. reed et al. compared the inhibitory effects of
phosphodiester and thiophosphate oligonucleotides comple -
mentary to bcl-2 mrnA on the growth of bcl-2-expressing
leukemia cells [68]. It turned out that a detectable inhibi-
tory effect of the phosphodiester asO n could be observed in
about half the time needed to develop a comparable effect
of the thiophosphate analog. However, the latter was more
effective in suppressing the growth of tumor cells. the thio-
phosphate asO n had the same effect as the phosphodiester
asOn at about a 5- to 10-time lower concentration [68]. It
was shown that an 80–95% reduction in Bcl-2 protein syn-
thesis after thiophosphate asOn treatment elevates the ap-
optosis rate and increases doxorubicin sensitivity [69].
Luzi and coauthors [184] developed a chemically modi-
fied ribozyme targeted at bcl-2 mrnA. the lipotransfection
of this drug into human raji lymphoma cells led to a 5-fold
drop in the bcl-2 mrnA level and to a 3-fold drop in the pro-
tein level, which was associated with a significant increase
in the proportion of apoptotic cells [184].
Fu et al. used bcl-2 sirnA to suppress Bcl-2 synthesis by
90% in cervical tumor cells HelaB2 and BGc-823, which led
to apoptosis induction [185].
In vivo. the decrease of the Bcl-2 protein level after treat-
ment with G3139 asO n, which was targeted at the respec -
tive gene’s m rnA, was associated with the suppression of
the oncogenic potential of lymphoma cells and a complete
blockage of tumor growth in mice [186]. the use of G3139 in
combination with cisplatin increased the anticancer chemo-
therapeutic effect by 70% [187].
recently, Morris et al. [188] reported the results of phase
I clinical trials of an 18-bp thiophosphate asOn G3139 (Ge-
nasense™, united States), which were complementary to
the first six codons of the bcl-2 open reading frame. Its was
shown that, after seven days of daily intravenous infusions
at a dose of 6.9 mg/kg, patients with non-Hodgkin lym-
phoma registered minor side effects, such as fatigue and
a reversible increase of transglutaminases in the blood se-
rum. Subcutaneous injection of the drug turned the cancer
process onto a stable, non-progressive mode in 9 out of 21
patients with non-Hodgkin lymphoma, and improved the
quality of life in 3 patients (a total objective response in 57%
of patients). In September 2000, the phase III trial of asO n
G3139 was launched on patients with chronic lymphatic
leukemia and acute myeloid leukemia [70]. the clinical trials
of this drug’s combinations with various chemotherapies for
melanoma and prostate carcinoma in patients resistant to
hormone therapy are now in progress [71, 72]. In February
2001, in 65 oncological clinics in the united States, canada,
and Great Britain, a phase III clinical trial of asO n G3139
was also launched for patients with multiple myeloma.
tumor-bearing mice, which recieved bcl-2 sirnA treat-
ment, displayed a retardation of liver tumor growth by
66.5% [185]. Mice with heterotransplants of pancreatic tu-
mors registered a 56% decrease in tumor volume [189].
Raf-1
In vitro. the 20-bp thiophosphate asOn ISIS 5132 oligo-
nucleotide is targeted at the 3′ -nontranslated region of c-
raf mrnA. this oligonucleotide effectively inhibited c-raf
mrnA accumulation and decreased the protein level in lung
carcinoma and colon and prostate tumor cells [190]. the ad-
dition of 2’-methoxyethyl modifications into the oligonucle -
otide asOn ISIS 13650 designed for c-raf suppression did
not lead to a significant increase of the inhibitory potential
[191]. In ovarian carcinoma cells, ISIS 5132 and ISIS 13650
induced 100% raf-1 suppression and an 80% drop in cell pro-
liferation [192]. Also, anti- c-raf asO n ISIS 5132 and ISIS
13650 were tested on 15 ovarian carcinoma cells lines. the
proliferation suppression efficacy varied from 10% to 90%.
Growth inhibition was associated with apoptosis and the
accumulation of cells in the G 2/M and S phases of the cell
cycle [193].
the comparison of c-raf suppression efficiency of asO n
and sirnA, which targeted the same region of m rnA, has
established that 125 nM of oligonucleotide caused a 52.4%
decrease in the raf-1 protein level, while the si rnA caused
only a 37.5% drop [194].

reVIeWS
№ 2 2009 | ActA nAturAe | 55
In vivo. Oligon ucleotide ISIS 5132 demonstrated a pro-
nounced antitumor effect in mice with ovarian carcinoma
heterotransplants [192]. the results obtained in in vitro and
in vivo experiments helped advance the studies of ISIS 5132
anticancer activity to clinical trials. A phase I trial demon-
strated that the drug was tolerated well by patients and
caused only a short period of thrombocytopenia in a few
cases [195]. A low toxicity treatment schedule was devised
which featured the slow intravenous infusion of the drug at
a daily dose of 2mg/kg in 21-day courses with a one week
break in between [195]. In the phase II clinical trial, the use
of this treatment scheme for patients with prostate cancer
[92], ovarian cancer [93], and colon adenocarcinoma [94]
demonstrated the stabilization of the cancer process in ap -
proximately 25% of the cases.
DNMT1
In vitro. the specific inhibition of the DnA-methyltrans-
ferase D nMt1 in cancer cells was achieved by the use of a
2’-О-methyl thiophosphate asO n MG98. the suppression
of DnMt1 by an asOn results in the demethylation of the
р16 gene promoter, the reestablishment of its activity, the
accumulation of cells with a hypomethylated retinoblas-
toma gene, and the inhibition of cancer cell proliferation
[107].
In vivo. In vivo experiments with asOn MG98 demonstrated
a significant growth retardation and regression of intestine
carcinoma and non-small cell lung cancer heterotransplants.
Successful preclinical studies insured the use of MG98 in
clinical trials [196]. the phase I clinical trial was focused on
evaluating the toxicity and establishing a pharmacological
profile of asO n MG98. Intravenous administration of the
drug at a daily dose of 80 mg/m 2 to patients with various
solid tumors for 21 days every 4 weeks proved to be rela-
tively safe [196]. However, no significant antitumor effect
was observed. Higher doses of the drug administered ac-
cording to the above-mentioned scheme were not tolerated
well by patients [196, 197]. In the phase II clinical trial, an
improved treatment scheme was tested on patients with
metastatic renal carcinoma. the dosage of 360 mg/m 2 was
administered twice a week for three weeks [108]. However,
this treatment schedule did not yield any positive response.
the authors argued that the clinical failure was due to inap-
propriate choice of tumor type [108].
RRR2
In vitro. the study of the asOn GtI-2040 targeted at rrr2
mrnA demonstrated a complete abrogation of rrr2 mrnA
accumulation in human lung carcinoma and significantly
decreased its amount in human bladder and murine fibro-
sarcoma tumors [116].
Duxbury et al. studied the ability of rrr2 sirnA to in-
crease the gemcytabine sensitivity of human pancreatic
adenocarcinoma cells implanted into mice via suppressing
the rrr2 subunit synthesis. Specific sirnA molecules tar-
geted at rrr2 mrnA significantly increased gemcytabine-
induced cytotoxicity [198].
In vivo. In vivo experiments demonstrated that anti-
rrr2 sirnA, in combination with gemcytabine, yield a syn-
ergistic antitumor antimethastatic effect [198].
the inhibitory potential of GtI-2040 was evaluated in
animal experiments. G tI-2040 suppressed tumor growth
in all experimental models, and the maximal effect was ob-
served in the renal carcinoma model, where a 95–98% re-
gression in the tumor was demonstrated [116]. A study of
asOn GtI-2040 in phase I clinical trials yielded a recom-
mended treatment schedule with the daily administration of
185 mg/m2/day for 21 days repeated every 4 weeks. Mono-
therapy according to this schedule did not cause any serious
side effects [117].
VEgF
In vitro. In vitro experiments demonstrate that a thiophos-
phate asOn targeting VEGF mrnA can reduce VeGF syn-
thesis by 45–83% in breast and bladder cancer cell lines
[124].
An rnAi approach was also assessed for the efficacy of
VEGF expression inhibition. Vector delivery of sirnA into
tumor cells caused the prolonged suppression of VeGF syn-
thesis by 33–53% [125].
In order to therapeutically interfere with pathological
angiogenesis, as an alternative strategy it was proposed
to target the VeGFr-1 and VeGFr-2 receptors, but not
VeGF itself. With this in mind, ribozymes complemen-
tary to regions of Flt-1(VeGF r-1) and KDR (VeGFr-2)
were designed [199]. It was shown that these ribozymes
specifically cleave rnA substrates in vitro and efficiently
decrease the mrnA level in cell cultures. currently, stud-
ies of the effect of D nAzymes on V eGFr-2 function are
being published. Zhang and coauthors demonstrate a 90%
decrease of V eGFr-2 protein level along with a 34–65%
drop in the viability of breast cancer cells due to apoptosis
induction [200].
In vivo. experiments in vivo demonstrated that VeGF inhi-
bition by thiophosphate asOn led to a 5-fold growth retar-
dation of renal carcinoma in mice [201].
In order to prolong the action and improve the drug per-
meability into the tumor cells, V eGF- targeted modified
sirnA were designed. An intratumor injection of cholesterol
oligoarginine-sirnA conjugate provided a 10-fold decrease
in the growth rate of colon adenocarcinoma [202], and in-
jecting an sirnA complex with a water-soluble lipopolymer
led to a 1.5-fold growth retardation of prostate adenocar-
cinoma [203]. In these experiments, a notable decrease in
tumor vascularization and reduction of V eGF expression
were also observed.
In preclinical trials, the Flt-1-targeted ribozyme exerted
antitumor, antiangiogenic and antimetastatic effects in a
metastatic lung cancer model [199]. Mice with grafted Lewis
lung carcinoma were surgically implanted with mini pumps
infusing the ribozyme at a rate of 12-100mg/kg daily. Af -
ter 19 days, the mini pumps were removed and new ones
installed. After treatment, a 92% tumor regression and a
70–80% reduction of lung metastasis were observed [199].
In mice with grafted intestinal carcinoma, the number of
metastases after treatment with anti-Flt-1 ribozyme de-
creased 3-fold [199]. this ribozyme was named Angiozyme
(ribozyme Pharmaceuticals, united States) and is now be-
ing evaluated in phase I and II clinical trials in patients with
various cancer types. Phase I trials were concluded in June

56 | ActA nAturAe | № 2 2009
reVIeWS
2001 and demonstrated good tolerance and disease stabiliza-
tion in 25% of patients [204]. Phase II trials in patients with
malignant forms of colon and breast cancer were aimed at
evaluating Angiozyme monotherapy and combinations with
traditional chemotherapeutic agents [35]. It was shown that
Angiozyme decreased the levels of soluble V eGFr-1 in se-
rum, but no significant clinical response was demonstrated.
these data underscore the importance of combining gene-
targeted and conventional chemotherapy. Promising results
were obtained in studies involving patients suffering from
colon tumors and featuring the simultaneous use of An -
giozyme with the Saltz therapeutic protocol (a combination
of bolus 5-fluoruracil, leucovorin, and irinotecan). this ap-
proach led to disease progression in only 12.5% of the cases,
as opposed to 25% in chemotherapy-only patients [35].
Interesting results were obtained in vivo with a
DnAzyme complementary to KDR (VeGFr-2) mrnA. Af-
ter four injections of anti-VeGFr2 DnAzyme, a 75% regres-
sion of breast cancer was observed [200]. the antiangiogenic
action of the D nAzyme reduced the vascularization of the
tumor tissue and therefore promoted cell death in the tumor
periphery.
neu/hER-2 (ErbB-2)
In vitro. An asOn complementary to Her-2/neu oncogene
was created and studied with respect to the antisense-me-
diated suppression of the gene in order to increase the effi-
ciency of conventional chemotherapy. In combination with
doxorubicin, the asOn additively suppressed tumor cell pro-
liferation [205].
the plasmid-encoded ribozyme complementary to neu
mrnA efficiently cleaved the rnA substrate in vitro. the
transfection of ovarian carcinoma cells by such a construct
caused a 50% reduction of the gene expression and a 39–42%
decrease in Her-2 protein level [131].
In vivo. AsOn targeting neu mrnA combined with doxoru-
bicin induced a synergistic anticancer effect [205].
the efficacy of Her-2 inhibition was assessed in vivo us-
ing injections of a recombinant adenovirus encoding a Her-
2 specific rybozyme [206]. three days after an intra-tumor
injection into subcutaneously grafted mammary cell carci-
noma, a 59% decrease of Her-2/neu mrnA was observged
in tumor tissue. treating such grafted mice with five weekly
injections resulted in 89% tumor regression [206]. Prelimi-
nary results of phase I trials of a Her-2/neu-targeted ribo-
zyme called Herzyme ( ribozyme Pharmaceuticals, united
States) in therapy-resistant mammary tumor patients dem-
onstrated disease stabilization but did not yield any cases of
a partial or complete positive response [132].
OThER TARgET gENES
Another asO n was designed to inhibit IGF-1 r activity. It
caused the mytocin-induced death of bladder carcinoma
cells in vitro [207], and the preincubation of melanoma
cells with this oligonucleotide prior to injection completely
blocked tumor graft development in mice [64].
the treatment of tumor cells with asO n targeted against
survivin helped increase the proportion of apoptotic cells [76,
77], and the use of anti-survivin ribozymes increased the sen-
sitivity of tumor cells to radio- and chemotherapy [79–80].
In in vitro experiments, the efficacy of drugs based on
asOn, si rnA, ribozymes and D nAzymes specifically for
suppressing B cr-ABL was demonstrated [84, 85, 87, 208,
209]. In vivo a thiophosphate asOn, complementary to Bcr-
ABL mrnA, proved to be most successful. the intravenous
injection of this drug prolonged the survival of mice with
developing leukemia [210].
to suppress the expression of the MDR1 gene and sensi-
tize the cells to cytostatics, the respective asOn [211, 212],
conjugate of asO n with doxorubicin [213], ribozyme [214],
and sirnAs targeted at different regions of the mrnA
were designed [215–218]. It was discovered that these
drugs greatly increased or reestablished in vitro tumor
cell sensitivity to doxorubicin [211, 213], vinblastin [215],
vincristin [214], daunorubicin [217], and paclitaxel [218]. In
vivo, the mice, which received subcutaneous grafts of colon
cancer cells expressing the anti-MD r1 ribozyme and were
later treated by doxorubicin, showed an almost complete
regression of hetereotransplant growth [98]. Our experi-
ments show that anti-mdr1b sirnA is effective in supress-
ing mdr1b expression in the cells of highly chemotherapy-
resistant rLS40 lymphosarcoma [219] and, when combined
with chemotherapy, specifically stimulates cell death in
vitro. If cyclophosphamide and anti-mdr1b sirnA are used
in combination in vivo, they cause the complete regression
of rLS40 lymphosarcoma in mice and increase the efficien-
cy of cyclophosphamide therapy more than 3-fold when
compared to cyclophosphamide monotherapy [99].
In order to circumvent MD r caused by the glutathione
system, a ribozyme targeted at the γ-glutamylcystein syn -
thetase was constructed. this drug increased the sensitivity
of colon tumor cells to cisplatin, doxorubicin, and etoposide
1.8-, 4.9- and 1.5-fold, respectively [101].
to overcome h tert-mediated tumor cell immortality,
specific h tert -targeted asO n were developed, such as
PnA, 2’-О-methyl asOn, (2-5А) asOn, which has a 2’5’-oli-
goadenilate group [111, 112]. 2’-О-methyl asO n and (2-5А)
asOn proved to be the most successful and caused the death
of 90% of the tumor cells in vitro [111, 112]. Daily intratu-
moral injections of (2-5А) asOn for 14 days caused a more
than 50% regression of a subcutaneous grafted glioma [112].
cells transfected by eIF4 e-targeted asO n decreased not
only the level of the factor itself, but also that of other can-
cer-associated proteins, such as Bcl-2, survivin, cyclin D1,
c-myc, and VeGF [136]. the intravenous infusion of anti-
eIF4e asOn to tumor-bearing mice caused a 10-fold regres-
sion of the tumor and did not have any toxic effect on the
animals’ healthy tissues and organs [136].
In order to suppress anomalous Ptn expression, a ri-
bozyme-based approach was suggested. In vivo experiments
showed that anti-Ptn ribozymes caused a more than 65%
retardation of melanoma growth in mice and inhibited an-
giogenesis by 70–85% [140]. Moreover, non-direct inhibi-
tion of the Ptn signaling pathway by ribozymes targeted at
ALK gene mrnA (which encodes a tyrosine kinase receptor
for Ptn) caused a 50–95% retardation of tumor growth and
a two-fold increase in the mean survival time for mice with
grafted glioblastoma [143].
Suppressing metalloproteinases by ribozymes did not
cause the complete cell death of tumors, but it retarded tu-

reVIeWS
№ 2 2009 | ActA nAturAe | 57
Table 3. Results of nucleic acid-based drugs testing in vivo
RNA-target Drug Tumor type Effect Trial stage
H-ras/K-ras
asOn
ISIS 2503
Hepatocellular carcinoma [162,
166]
Decreased tumor weight in mice and suppressed metastatic proc-
cesses [162, 166]. Low toxicity, optimized therapy schedule [48,
168], positive response in combination with gemcytabine in 10% of
patients; one complete response, 4 partial [48]
Phase II
ribozyme throat, pancreatic and bladder
carcinoma, melanoma
Suppresses mouse tumor growth as a result of an increased pro-
portion of apoptotic tumor cells, decreases the invasive potential
of the tumor, and increases animal survival time [43–45, 169]
In vivo
sirnA pancreatic and ovarian carcinoma
[163, 164], lung cancer [165] Inhibition of tumor growth [163–165] In vivo
c-myc
asOn
AVI-4126
(AVIBioPharma, uSA)
Prostate and breast cancer
[50, 51]
Suppresses tumor growth by 80% in mice [50].
Moderate toxicity, accumulation of the drug in tumor tissue [51] Phase II
sirnA breast cancer [177],
lymphoma [176]
Opposes tumor progression [177]; decreases с-myc mrnA level in
serum down to 15–20% [176] In vivo
PKc-α
asOn
ISIS 3521
(AffinitaktM, uSA)
Bladder carcinoma, lung and
colon cancer [55], ovarian cancer
[181], colon carcinoma [182]
completely suppresses tumor growth in mice at a dosage of ISIS
3521 of 0.06–0.6 mg/kg [55].
Moderate toxicity in clinical trial [180]; 3 objective responses to
therapy [181]; no response to therapy [182]
Phase II
clusterin asOn
OGX-001
renal carcinoma [59], prostate
cancer [60]
caused a 2-fold decrease in tumor volume in mice when used in
combination with paclitaxel [62].
Decreased clusterin levels in the patients’ pathological tissues [60]
Phase I/II
IGF-1r asOn Melanoma completely blocked tumor development from cells transfected
with the asOn [64] Ex vivo
bcl-2
asOn G3139
(Genasense™, uSA)
Lymphomas, lymphatic
leukemia, myeloleukemia [186,
187], melanoma [71], lymphoma
[188], lympholeucosis [70],
prostate carcinoma [72]
Decreased tumor volume in mice [186], additive antitumor effect
of asOn G3139 and cisplatin [187].
Moderate toxicity in clinical trial, stabilized the tumor process,
improved life quality [188]
Phase III
sirnA Murine liver Н22 tumor [185],
pancreatic cancer [189]
Slowed liver tumor growth in mice by 66.5% [185]; decreased
pancreatic cancer heterotransplant volume by 56% [189] In vivo
bcr-abl asOn chronic myeloleukemia 2-fold increase of mice mean survival time [210] In vivo
c-raf
asOn
ISIS 5132
(neopharm, uSA)
Ovarian [93, 192] prostate can-
cer [92], colon adenocarcinoma
[94]
Suppressed tumor growth in mice [192].
Moderate toxicity in clinic [195], stabilization of the disease in
more than 25% of cases [92, 93]
Phase II
MDr1, mdr1a/
mdr1b
ribozyme colon adenocarcinoma A virtually complete regression of mouse tumors in an ex vivo
experiment, in combination with doxorubicin [98] Ex vivo
sirnA Murine lymphosarcoma [99,
219]
A virtually complete regression of mouse tumors in an in vivo
experiment, in combination with cyclophosphamide [102] In vivo
DnMt1 asOn MG98
non-small cell lung cancer,
colon carcinoma, metastatic
renal carcinoma and other solid
tumors [108, 196, 197]
regression of tumors in nude mice [196].
no serious side effects, no clinical response from patients
[108,196, 197]
Phase II
htert asOn Glioma A more than 50% regression of tumor [112] In vivo
rrr2
asOn
GtI-2040 (Genasense™,
uSA)
Solid tumors
Inhibited tumor growth in all the experimental models studied,
maximal effect in renal carcinoma. 95–98% regression [116].
no serious side effects, optimized treatment schedule [117]
Phase II
sirnA Pancreatic adenocarcinoma Synergistic sirnA and hemcytabine cytotoxicity [198] In vivo
VEGF, Flt-1
(VeGFr1),
KDR (VeGFr2)
asOn renal carcinoma Fivefold tumor regression [201] In vivo
sirnA colon and prostate adenocarci-
noma [202, 203]
tenfold retardation of colon adenocarcinoma growth [202] and
1.5-fold inhibition for prostate carcinoma [203], suppresses tumor
vascularization [202, 203]
In vivo
ribozyme
Angiozyme, Sirna ther.,
uSA
Lung carcinoma [199], colon
cancer [35, 204], breast cancer
[35]
Growth regression of Lewis lung carcinoma in mice by 92% and
70–80% decrease in metastasis in the lungs [199].
Well-tolerated by patients, decreases VeGFr-1 protein level in
tumor cells, stabilizes the disease in 25% of patients [35, 204]
Phase II
DnAzyme Breast cancer
causes tumor regression by 75% in mice, reduces vasularization
in tumors, and causes cell death in the tumor’s peripherial tissue
[200]
In vivo
neu (Her-2/
erbB2)
asOn Ovarian and breast cancer Synergistic anticancer effect with doxyrubicin [205] In vivo
ribozyme
Herzyme Breast carcinoma regression of tumors in mice by 89% [206].
Well-tolerated by patients, stabilizes the disease [132] Phase I
eIF4e asOn
LY2275796 Prostate carcinoma tenfold regression of the tumor; has no toxic effect on healthy
organs or tissues in mice [136]
PTN и ALK
ribozyme
(anti-Ptn) Melanoma reduces tumor size by 65%, inhibits vascularization by 70–85%,
and induces apoptosis [140] In vivo
ribozyme
(anti-ALK) Glioblastoma Delays tumor growth, increases tumor-bearing mice survival time
2-fold [143] In vivo
MMP9 ribozyme Metastatic fibrobalsts [220],
prostate cancer[147]
causes an 8-fold decrease in metastasis, a 1/3 increase in mean
mouse survival time, and has no effect on primary tumor develop-
ment [147, 220]
Ex vivo
FGF-BP ribozyme Prostate carcinoma Suppresses tumor development [152] Ex vivo
eGr-1 DnAzyme Breast carcinoma Suppresses tumor growth 3-fold [157] In vivo
FAK sirnA Prostate and breast carcinoma tumor regression [158] In vivo
cXcr4 sirnA Breast carcinoma Virtually completely inhibits metastasis, decreases CXCR4 mrnA
level to 10% [159], and causes tumor regression [223] In vivo

58 | ActA nAturAe | № 2 2009
reVIeWS
mor growth, limited its volume increase, and suppressed
angiogenesis and metastasis [147, 220, 221].
A ribozyme targeted at FGF-BP causes an 80% suppres-
sion of the proteins synthesis which inhibited cell prolifer-
ation in prostate and colon carcinomas [152, 222]. tumors
transfected by an anti-FGF-BP ribozyme and later subcuta-
neously grafted into mice did not show any signs of progres -
sive development [152].
In order to suppress eGr-1, a DnAzyme was constructed.
It targeted the mrnA of the transcription factor eGr-1. In-
tratumoral injections of this drug caused a 3-fold decrease
in the tumor's size [157].
Specific si rnA was developed to suppress FAK hyper
function in the cells of prostate and mammary glands. cells
transfected by this si rnA demonstrated the inhibition of
cell adhesion, migration, and proliferation in vitro and tu-
mor growth suppression in vivo in mice [158].
In vivo experiments showed that intravenous injections
of anti-cXcr4 sirnA not only decreased the CXCR4 mrnA
level to 10% of the control level, but also virtually blocked
metastasis into the lungs [159] and suppressed the primary
tumor growth [223].
using drugs based on nucleic acids to fight cancer is one
of the most promising and rapidly developing areas of mod-
ern molecular oncology. Gene-targeted oligonucleotides
make it possible to inhibit key links in the malignant growth
process. these drugs can not only help regulate prolifera -
tion, apoptosis, and drug resistance, but they also affect the
cell–cell interactions that promote malignant progression
in the entire organism. However, it is worth noting that, al-
though gene-directed oligonucleotides share the mechanism
of action (which involves switching off the target gene), the
efficiency of drugs directed at suppressing the same gene,
even through the same region in the mrnA target, can vary
significantly.
Some companies have already undertaken the develop-
ment and marketing of anticancer drugs based on gene-tar-
geted oligonucleotides, such as asOn and ribozymes, which
are well recognized as forerunners of the gene-targeted
therapeutic approach. As is evident from table 3, the genes
most sensitive to asOn turned out to be involved in prolif-
eration and apoptosis blockage in tumor cells. Also, genes
like Flt-1 (VeGFr) and neu (Her-2) turned out to be very
susceptible to the effect of ribozymes. AsO n targeted at
suppressing H-ras, c-myc, PKC-α , clusterin, bcl-2, c-raf,
and ribozymes, which cleave the Flt-1 and neu mrnA, are
currently being evaluated in clinical trials. certain types of
gene-targeted nucleic acid drugs such as DnAzymes and
sirnA have only recently started to be seen as promising
therapeutic tools; however, the amount of successful clini-
cal trials is rapidly catching up with asO n and ribozymes.
Drugs for curing age-related retinal degeneration based on
sirnA are currently being tested in clinical trials. However,
sirnA for treating cancer have not been introduced into
clinical trials yet.
Despite the undoubted achievements of modern oncol -
ogy, the problem of raising the efficiency of malignant neo-
plasia treatment is still very pressing. notably, one of the
main objectives of gene-targeted therapy is to provide the
delivery of drugs to the target cells, which presumes the
transport into a specific organ or tissue, traversing the cel-
lular membrane, and arriving at specific cell compartments.
It is clear that oligonucleotides cannot do this by themselves.
therefore, the development of vehicle systems which could
increase the efficiency of these drugs is of utmost impor-
tance. the problem of transporting a specific rnA or D nA
sequence into a tumor cell is still unsolved.
As was noted above, changes in the expression of cer-
tain genes play a key role in oncogenesis. t he dysfunction of
these genes can affect the correct regulation of the signaling
pathways that control transitions between the phases of the
cell cycle, proliferation, apoptosis, genetic stability, differ-
entiation, and morphogenetic reactions of the cell. these
processes in the cell are tightly connected and are often in-
terchangeable. In many cases, drugs based on nucleic acids
affect a specific mechanism, which leads to the abrogation
of a whole chain of malignant growth regulation. However,
it is important to bear in mind that one of the most crucial
properties of tumor cells is the ability to exploit cell sur-
vival pathways, which allows these cells to escape gene-
targeted molecular control. this consideration, however,
does not undermine the importance of cancer therapy using
nucleic acid-based drugs directed at a specific regulatory
link. nevertheless, the flexibility and plasticity of the bio-
chemical profile, as well as the robustness of the regulation
of functions vital for the survival of tumor cells, require
that gene-targeted cancer therapy be optimized as much
as possible.

reVIeWS
№ 2 2009 | ActA nAturAe | 59
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