Delivery Systems for In Vivo Use of Nucleic Acid Drugs

REVIEW
Delivery Systems for In Vivo Use of Nucleic Acid Drugs
Resende R.R1,4, Torres H.A.M2, Yuahasi K.K3, Majumder P1 and Ulrich H1
1Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo
05508-900, SP, Brazil. 2Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo
04023-062, SP, Brazil. 3Departamento de Neurologia e Neurocirurgia, Universidade Federal de São
Paulo, São Paulo, SP, Brazil. Present address 4Departamento de Fisiologia e Biofísica, Instituto de
Ciências Biomédicas, Universidade de São Paulo, 05508-900 São Paulo, SP, Brazil.
Abstract: The notorious biotechnological advance of the last few decades has allowed the development of experimental
methods for understanding molecular mechanisms of genes and new therapeutic approaches. Gene therapy is maturing into
a viable, practical method with the potential to cure a variety of human illnesses. Some nucleic-acid-based drugs are now
available for controlling the progression of genetic diseases by inhibiting gene expression or the activity of their gene prod-
ucts. New therapeutic strategies employ a wide range of molecular tools such as bacterial plasmids containing transgenic
inserts, RNA interference and aptamers. A nucleic-acid based constitution confers a lower immunogenic potential and as
result of the high stringency selection of large molecular variety, these drugs have high affi nity and selectivity for their
targets. However, nucleic acids have poor biostability thus requiring chemical modifi cations and delivery systems to main-
tain their activity and ease their cellular internalization. This review discusses some of the mechanisms of action and the
application of therapies based on nucleic acids such as aptamers and RNA interference as well as platforms for cellular
uptake and intracellular delivery of therapeutic oligonucleotides and their trade-offs.
Keywords: aptamers, RNA interference (RNAi), drug delivery systems, nucleic-acid-based drugs.
Introduction
The discovery of the DNA molecule was one of the most important achievements in the understanding
of the fundamental basis of life and now its untapped therapeutic potential is being revealed. Therapies
based on nucleic acids (NAs) including plasmids containing transgenes used in gene therapy, antisense
and antigene oligonucleotides, ribozymes, DNAzymes, DNA and RNA aptamers and small interfering
RNAs, have been developed over the past couple of decades (Crooke, 1998; Stull and Szoka, 1995;
Patil et al. 2005; Ulrich et al. 2006). Although most NA-based drugs are in the early stages of clinical
trials, this molecule class has emerged during recent years as promising drug candidates able to act in
a large range of diseases including AIDS, cancer and neurological and cardiovascular disorders (Stull
and Szoka, 1995; Patil et al. 2005; Ulrich et al. 2006). The sequencing of the human genome and the
transcriptome and proteome projects are providing additional platforms for the advancement of NA-
based therapies by supplying new targets for the design, screening and selection of drugs. One of the
most signifi cant advantages of NA-based drugs over conventional pharmaceutical drugs is its high
selectivity towards its molecular targets resulting in very specifi c physiological action. These drugs can
be used to investigate the genetic disease condition or used in prophylactic measures, thereby preventing
disease progression and/or complications in its early stages. For instance, gene therapy usually involves
the correction of a malfunctioning gene by the introduction and expression of its correct copy, thus
resulting in a corrected protein product. Similarly, NA-based drugs involved in gene ablation turn off
only selected genes, guaranteeing specifi c control of the disease state. Thus, at least theoretically, the
use of NA-based therapies can result in null or minimal collateral effects when compared to conven-
tional, often less specifi c, pharmaceutical drugs.
However, the effects of NA drugs on human exposition must be completely understood, with
emphasis to unforeseen long-term effects. There is yet little knowledge about pharmacokinetics of
NA-based drugs. The greatest challenge facing the therapeutic utility of NAs is to overcome the
low cellular absorption inherent to their highly polar molecular structure. The innate ability of these
drugs to cross membranes is minimal in normal circumstances. In addition, their low biostability
Correspondence: Henning Ulrich, Departamento de Bioquímica, Instituto de Química, Universidade de São
Paulo, Av. Prof. Lineu Prestes 748, 05508-900, São Paulo, SP, Brazil. Tel: + 55-11-3091-3810, Ext. 223;
Email: henning@iq.usp.br
Please note that this article may not be used for commercial purposes. For further information please refer to the copyright
statement at http://www.la-press.com/copyright.htm
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results in unpredictable pharmacokinetics. NA (si)-RNAs (products of the long dsRNA cleavage)
molecules that happen to enter the cell are subse-
are integrated into the RISC complex (RNA-
quently subjected to intracellular degradation by induced silencing complex). The complex
nucleases effectively narrowing the drug’s becomes activated by the unwinding of the duplex
activity time-span.
upon the loss of one strand of the si-RNA
The fi rst NA-drug approved in November 1998 duplex by an RNA helicase activity. Depending
by the FDA was denominated Vitravene®, an oligo-
on several target and si-RNA properties that are
nucleotide discovered by ISIS® as antisense drug not entirely understood, RISC can either specifi -
for the treatment of cytomegalovirus retinitis in cally cleave and degrade target mRNA (Yekta
AIDS patients (Perry and Balfour, 1999). Vitravene® et al. 2004; Meister et al. 2004; Zamore et al.
was marketed by Novartis Ophthalmics® and 2000; Bagga et al. 2005; Giraldez et al. 2005;
revealed to be a powerful AIDS medication. In Wu et al. 2006) or inhibit its translation without
preclinical studies, antisense inhibition of c-Raf initiating its sequence-specifi c mRNA degrada-
kinase was associated with a reduction in the tion process (Olsen and Ambros, 1999; Reinhart
formation of new blood vessels in the eye involved et al. 2000; Wightman et al. 1993). The target
in both, age-related macular degeneration (AMD) mRNA is cleaved by the RISC complex at the
and diabetic retinopathy (Danis et al. 2003). The middle of the complementary region, ten nucleo-
approval of this fi rst nucleic-acid based drug serves tides upstream of the nucleotide paired with the 5'
as an encouragement for scientists to further use end of the guide siRNA (Elbashir et al. 2001b).
SELEX Systematic Evolution of Ligands by Expo-
The cleavage reaction guided by RISC does not
nential Enrichment and RNA interference (RNAi) require ATP (Nykänen et al. 2001; Hutvágner and
approaches for drug development (see Fig.1A for Zamore, 2002). However, multiple rounds of
a scheme of oligonucleotide action on gene expres-
mRNA cleavage, which requires the release of
sion and protein acivity).
cleaved mRNA products, are more effi cient in the
presence of ATP (Hutvágner and Zamore, 2002).
Nucleic-acid Based Therapies
When the dsRNA is of endogenous origin, the
Dicer cleavage products are named micro RNA
Mechanisms of action small interfering (miRNA). The miRNA-guided mechanism of
RNAs
translational regulation is not as well understood.
Artifi cial modulation of gene expression is mainly Studies of mutant or transgenic C. elegans,
based on the inhibition of gene transcription or showed that miRNAs inhibited target-protein
mRNA degradation. The phenomenon of RNAi synthesis without affecting mRNA levels (Bartel,
was first observed in the nematode worm 2004). The target mRNA contains three-prime
Caenorhabditis elegans in response to a double-
untranslated regions with several binding sites for
stranded RNA (dsRNA) treatment which resulted the miRNA, and both the target and the miRNA
in sequence-specifi c gene silencing (Fire et al. were found to be associated with polyribosomes.
1998). Later, the same phenomenon was observed This suggested that miRNAs block translation
in a large variety of biological systems including elongation or termination rather than translational
several invertebrates and, more recently, also initiation (Olsen and Ambros, 1999; Seggerson
vertebrates such as Xenopus and mice (Nakano et al. 2002).
et al. 2000; Wianny and Zernicka-Goetz, 2000).
By using protein mutants various genes involved
For induction of RNAi, small double stranded in the RNAi process were identifi ed, including
RNAs (termed small interfering RNAs – siRNAs) some highly homologous helicases and other
are produced by the cleavage of long dsRNAs enzymes involved in the transposition of mobile
(Tuschl et al. 1999; Zamore et al. 2000; Hamilton elements, such as RNase D (Bertrand et al. 2002;
and Baulcombe, 1999; Hammond et al. 2000). McManus and Sharp, 2002; Scherr et al. 2003).
The cytoplasmic, highly conserved Dicer protein, Results like these corroborate the currently
member of the family of RNase III–like enzymes, accepted hypothesis that the response to dsRNAs
forms a characteristical 21–23-nucleotide long plays a defensive physiologic role against delete-
dsRNA duplex with symmetric two- to three-
rious RNAs such as virus transcripts. Therefore
nucleotide 3' overhangs (Bernstein et al. 2001; miRNAs are essential for the maintenance of
Elbashir et al. 2001a). The duplex small-interfering genome integrity in a large range of biological
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Delivery systems of nucleic acids drugs
Figure 1. Nucleic-acid based therapies. (A) Actions of antisense oligonucleotides, small interfering RNAs, aptamers and intramers (intracel-
lularly acting aptamers) on target cells. The antisense technology is based on the introduction of a complementary oligonucleotide sequence
to the target mRNA resulting in RNAse H activation and target RNA degradation. For RNA interference process 21–23 nucleotide-long
siRNAs induced into the cell activate the RISC complex leading to degradation of target mRNA. RNA or DNA aptamers target and inhibit
the products of gene expression such as intracellular or extracellular protein. (B) Conventional delivery of siRNA to target cells by transfec-
tion. Therapeutic applications are hindered due to poor cellular uptake and absence of a mechanism to deliver siRNAs specifi cally to target
cells. (C) Aptamer-directed siRNA delivery to target cells. The si-RNA is coupled to an aptamer which specifically binds a surface epitope
on target cells (i.e. prostate-specifi c membrane antigen), thereby possibiliting the specific down-regulation of gene expression in cancer
cells. Following binding of the siRNA-aptamer chimera to its cell-surface receptor, the receptor-oligonucleotide complex is internalized fol-
lowed by induction of RNA interference.
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Ulrich et al
systems (Hutvágner and Zamore, 2005). The Cand6 which also suppresses VEGF gene expression
interference effect of dsRNA is non-stoichiometric is currently being tested in a phase 2 trial with
in relation to homologous mRNA, since very low patients suffering from AMD. Moreover, early in
amounts of dsRNA cause strong interference. This 2006, Cand5 was also tested in phase 2 clinical
suggests that the RNAi effect involves a catalytic trials for the treatment of diabetic macular edema
stage and cannot be based on the titration of endog-
(http://www.acuitypharma.com/). The siRNA
enous mRNA (Sverdlov, 2001).
ALN-RSV01 developed by Alnylam Pharmaceu-
ticals, Inc., for treatment of RSV infection has
Therapeutical applications of RNAi
completed two phase 1 trials and now appears to
Currently, siRNA drugs are being developed in be appropriate for tests in humans. Other siRNAs
order to inhibit cell infection by HIV (Martinez targeting Influenza and hepatitis C viruses
et al. 2002) and Infl uenza viruses (Ge et al. 2003) (Protiva Biotherapeutics) are expected to be avail-
and to treat autoimmune hepatitis (Song et al. 2003) able for clinical use within the coming year
(Fig. 1B). RNAi has also been used as a tool to study (Protiva Biotherapeutics).
signaling pathways involved in neurogenesis and
neurodegeneration (Miller et al. 2005). As an Stability of siRNA in plasma
example, the function of vascular endothelial siRNA molecules are unstable in serum as a result
growth factor (VEGF) in directing neurogenesis of degradation by serum nucleases and thus have
was verifi ed via RNAi (Cao et al. 2004). Another very short half-lives in vivo (Soutschek et al. 2004).
approach uses adenoviral, lentiviral and AAV Stability against nuclease degradation can be
(adeno-associated viral) delivery systems (Chen achieved by introducing a phosphorothioate (P = S)
et al. 2006) to treat spinocebellar ataxia (Xia et al. backbone linkage at the 3′ end for exonuclease
2004), amyotrophic lateral sclerosis (ALS) (Ralph resistance and 2′modifi cations (2′-OMe, 2′-F or
et al. 2005; Raoul et al. 2005), Huntington´s related) for endonuclease resistance (Vornlocher
(Harper et al. 2005; Rodriguez-Lebron et al. 2006) et al. 2005; Li et al. 2005; Choung et al. 2006).
and Alzheimer’s disease (Singer et al. 2005).
Moreover, siRNA molecules consisting entirely of
When compared to other gene ablation tools 2′-O-methyl and 2′-fl uoro-modifi ed nucleotides
such as antinsense oligonucleotides, siRNAs are demonstrated enhanced plasma stability and
notably superior due to their higher degree of increased in vitro potency. Duplexes containing
mRNA degradation and low potential of inducting the 4′-thioribose modifi cation present increased
immune responses (Bertrand et al. 2002). Since thermal stability and are 600 fold more resistant to
siRNAs molecules are not integrated in the genome degradation in plasma than natural RNA duplexes
as plasmids potentially are, they cause collateral are (Hoshika et al. 2004). Substantial improve-
effects and thus are much more therapeutically ments in siRNA activity and plasma stability have
advantageous. Small-interfering RNAs do not need also been achieved by judicious combination of
to be transferred into the nucleus in order to present 4′-thioribose with 2′-O-Me and 2′-O-methoxyethyl
activity, requiring less sophisticated delivery modifi cations (Dande et al. 2006).
systems. In addition, due to their relative small
size, the delivery of a cocktail of siRNAs targeting
the expression of multiple disease causing genes Aptamers
at the same time should be feasible.
Aptamers are oligonucleotides identifi ed by an
VEGF gene expression was the fi rst target for in vitro selection process as high-affi nity binders to
clinical trials using siRNA in the treatment of a given target molecule. For this purpose, a DNA
age-related macular degeneration (AMD). RNAi library is synthesized containing an inner random-
is also being used to combat infection by the ized sequence of typically 20–100 nucleotides
respiratory syncytial virus (RSV) genome. The fl anked by two outer constant regions of 20–40
vehicle used in both cases was a saline formula-
nucleotides. A T7 promoter site is incorporated in
tion. The success of these trials may be due to the one of the constant sequences, if a RNA aptamer is
direct administration of siRNAs at the diseased to be selected. The chemically synthesized DNA
organs. The advantage of the direct administration pool is amplified by PCR in the presence of
is the high concentrations of the siRNA available sense- and anti-sense-primers. The DNA template
at the target side. A siRNA-based drug denominated can now either be transcribed in vitro to the RNA
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Delivery systems of nucleic acids drugs
pool or be denatured to originate a single-stranded survival in the presence of the aptamer indicated
(ss) DNA pool to be used in the in vitro selection crucial functions of the B52 protein (Shi et al.
process. In many cases 2′-F-modifi ed pyrimidines 1999). Another approach in therapeutics makes use
are employed in the in vitro transcription reaction of an aptamer that binds the intracellular domain
to improve nuclease-resistance of generated RNA of the β−2 integrin lymphocyte function-associated
molecules (reviewed by Ulrich et al. 2004). Re-
antigen-1 (LFA-1). This portion mediates cell
iterative cycles of in vitro selection, also denomi-
adhesion by binding to the intercellular adhesion
nated as systematic evolution of ligands by expo-
molecule-1 (ICAM-1). The specifi c blockage of
nential enrichment (SELEX), are carried out by signaling pathways in vivo by intramers could
incubating the target protein or another molecule of potentially be applied to any signal-transduction
biological importance with the combinatorial DNA cascade (Blind et al. 1999). The intramer against
or RNA pool, followed by elution and amplifi cation the rev protein, which is involved in the cycle of
of target binders by RT-PCR or PCR techniques. replication of HIV resulted in inhibition of the virus
Selection stringency is increased with the numbers replication in cell culture (Good et al. 1997), and
of SELEX cycles. Increased stringency can be in human lymphocyte cells (Chaloin et al. 2002).
achieved by augmenting the number of DNA or These results indicate that intramers may be an
RNA molecules relative to possible target binding alternative to RNAi by specifi cally suppressing the
sites as well as by extensive washing for removal activity of gene products instead of inducing degra-
of low-affi nity binders. These procedures ensure dation of mRNAs coding for these proteins.
that the original random pool containing 1013–1015
The ability to modulate intramer activity would
different sequences becomes narrowed down to a drastically increase the effi ciency of regulation
more homogenous population of high-affi nity target of intracellular signaling by oligonucleotides. The
binders. When the binding affi nity of a selected RNA activity of these aptamers could be put under the
or DNA library to its target cannot be any longer allosteric control of a second molecule (Tang and
improved by subsequent SELEX cycles, this fi nal Breaker, 1997) or their expression could be allo-
pool is sequenced for identifi cation of aptamers. At sterically regulated. The sequence coding for the
this stage one expects that similar sequence motifs intramer can be introduced in the 5′-promoter
have been preserved in aptamers with binding position and work as an inductive promoter. Some
affi nity to their targets and that in most cases these intramers were developed against kanamicin and
consensus sequences fall into conserved stem-loop tobramicin and expressed in the position of the
motifs. These RNA or DNA molecules with unique 5′-gene promoter. The addition of those antibi-
binding characteristics, also denominated as otics to the cell system resulted in the shut down
aptamers (from latin aptus = to fi t) can be used for of the 5′-gene transcription (Werstuck and Green,
basic research, clinical and diagnostic purposes. 1998).
Basic research purposes include the characterization
The high specifi city of aptamers in acting just
of aptamer-target protein interaction in its cellular on an isotype or a splice-variant of a target protein
context (Ulrich et al. 1998, 2002), the study of the makes them excellent drug candidates. In this
mechanism of protein activation and inactivation regard, Theis and colleagues identifi ed intramers
(Hess et al. 2000), the mapping of binding sites (Shi which bind and switch off the cytohesin-2 guanine
et al. 2007), the dissection of intracellular signaling nucleotide exchanger but do not affect the homol-
pathways (Famulok et al. 2001) as well as the inhi-
ogous protein cytohesin-1 that has a different
bition of intracellular virus replication (Toulmé et al. function. This effect was observed following six
2003).
hours of HeLa cell transfection with the aptamer
An example for an intracellular acting aptamer construct (Theis et al. 2004). These properties of
(intramer) is an RNA molecule selected as high-
intramers make them promising tools for develop-
affi nity ligand of the B52 protein. The protein B52 ment of therapeutics with applications in vaccine
is expressed during Drosophila development and development, blockage of intracellular transduc-
acts there in the gene-splicing process. As the exact tion pathways, in viral infection control and time-
function of this protein was not fully understood, dependent gene knockdown of protein activity.
an anti-B52 intramer was developed and expressed The suppression of target protein activity at
as a pentameric structure in developing Drosophila desired time points followed by aptamer inactiva-
cells. The observed drastic reduction of Drosophila tion may gain importance in the control of important
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Ulrich et al
physiological functions such as blood coagulation et al. 2004). These modifi cations in oligonucleotide
(Rusconi et al. 2004). Inhibition of coagulation structure can be done prior or following the SELEX
is for instance desirable in a pathological condi-
process. (III) Identifi ed aptamer sequences can be
tion of high blood pressure, but must be immedi-
truncated to their minimal sequences, represented
ately reversed in case of hemorrhage. Moreover, by a single loop structure which is suffi cient for
aptamers are very potent inhibitors of extracel-
binding and biological activity. (IV) Aptamers can
lular protein activity, for instance by blocking be easily enzymatically reproduced or produced in
growth factor-receptor binding. An anti-VEGF
a large scale by chemical synthesis (reviewed by
165
aptamer was developed that blocks pathological Ulrich et al. 2006).
VEGF165-receptor binding, thereby leaving other
vital VEGF isoform action unaffected. An anti-
VEGF
Vehicles for Oligonucleotide
165 aptamer formulation has been recently
approved for therapeutic use by the FDA and Delivery
trade-named as Macugen (reviewed by Ulrich et The combination of target-specifi c drug delivery
al. 2006; Vavvas and D’Amico, 2006; Ng et al. and its controlled release (Langer, 1998) is an
2006).
important goal in the search for more effi cient and
Another promising aptamer tested in clinical less hazardous treatment of tumoral diseases. It is
trials has been denominated REG1 (Regado Biosci-
desirable that the cytotoxic drug dosage is delivered
ences). This therapeutic aptamer targets factor IXa to target cells for a long time span, thereby sparing
(drug, RB006) with anti-coagulation activity. The the healthy cells of the surrounding tissue. In order
aptamer and its complementary oligonucleotide to attain this objective, it is critical to develop
antidote (RB007) were tested in healthy volunteers specialized vessels which encapsulate the chemo-
in a Phase 1A Pharmacodynamic Evaluation (Dyke therapeutical drugs for its controlled release and
et al. 2006). There was not any signifi cant bleeding that such vessels are directed to cancer cells (i.e.
occurrence associated with RB006 treatment, and by presenting appropriate ligands which recognize
both aptamer drug and antidote were well tolerated. specifi c cancer-cell antigens).
The overall results of the pharmacokinetics of the
A wide variety of address-molecules have been
two compounds in healthy volunteers indicated investigated for their effi ciency to deliver oligo-
their safe use in humans encouraging further nucleotides to cancer cells. Humanized antibodies
studies (Dyke et al. 2006).
and single chain variable fragments generated by
In addition to their possible therapeutic rele-
murine hybridomas or phage displayed, minibodies
vance, aptamers may be used in diagnostic applica-
and peptides were among the tested delivery
tions, such as differentiating between normal and vehicles (reviewed by Weiner and Adams, 2000).
tumoral vasculature (Blank et al. 2001), the patho-
For the development of vehicles, some pre-requisites
genic form of the prion protein from its normal must be satisfi ed in order to improve their chances
conformation (Rhie et al. 2003), as well as identi-
of passing functional and clinical trials. The drug
fying possible biohazards, such as Anthrax spores encapsulating particle-system must be composed
(Bruno and Kiel, 1999). The possible pharmaceu-
of biocompatible, biodegradable polymers
tical and therapeutic importance of aptamers is approved for clinical use by the drug regulating
mainly related to the following characteristics: (I) agencies. Moreover, the vessel particles must
their affi nity for their targets with dissociation effi ciently bind the negative charges of NA chains
constants in the nano- to picomolar range, similar while minimally adverse-effecting their tridimen-
to those found on monoclonal antibody-antigen sional folding and thus their binding properties.
complexes. High-affi nity aptamer-protein interac-
The delivery vessels must yet effi ciently and selec-
tions result from specifi c hydrogen-bond formation tively bind to target cells, as well as have a long
between bases and amino acids of the target half-life in circulation in order to reach the target
proteins in addition to interactions between oligo-
before being degraded and releasing their contents.
nucleotide backbones and protein secondary and Vehicles may be classifi ed as such (I) improving
tertiary structures. (II) Aptamers can be chemically oligonucleotide pharmacokinetics by attaching a
modified to acquire more stability for in vivo high-molecular weight lipophilic molecule to an
applicatons, resulting in an increase of their half-
aptamer in order to augment the half-life of a
life time from a couple of seconds to days (Ulrich therapeutic oligonucleotide in the plasma, and as
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Delivery systems of nucleic acids drugs
such (II) permitting the immobilized oligonucleotide cell or tissue as therapeutical nanoparticle
to pass physiological barriers such as the brain-
is the most desirable aim of any delivery system.
blood barrier or plasma membranes to be delivered Previously selected RNA aptamers specifi cally
into cells. In addition to their ability to act as binding to PSMA were used to escort Gel to
therapeutic drugs by themselves, aptamers can also tumoral cells expressing PSMA at their surface.
be used as vehicles themselves to deliver another The conjugated toxin destroyed prostate cancer
oligonucleotides to specifi c target cells (Farokhzad cells with an IC50 value of 27 nM, presenting an
et al. 2006).
increase in toxicity of more than 600 times in
comparison to cells which do not express PSMA
(Chu et al. 2006). The extracellular domain of
Applications for oligonucleotide-
PSMA can now be recognized by a biocompatible
directed drug delivery
and biodegradable polymeric nanoparticle encap-
Prostate-specifi c membrane antigen (PSMA) is a sulated with a docetaxel (Dtxl) surface function-
type-2 integral membrane glycoprotein, expressed alized with a stable, nuclease-resistant RNA
at the prostate carcinoma surface and on new aptamer containing 2′-F-modified pyrimidine
vessels formed by various other solid tumors. bases (Farokhzad et al. 2006). These aptamer-
This antigen is highly expressed in every stage nanoparticle bioconjugates (Dtxl-NP-Apt) bound
of prostate cancer development (Rajasekaran et al. to PSMA proteins expressed at the surface of
2005) and, therefore, is a strong molecular target prostate epithelial cells LNCaP were easily incor-
candidate for immunotherapy and prostate-cancer porated by cancer cells with cytotoxic effects in
imaging. Previous efforts to selectively destroy vitro. Encouragingly, Dtxl-NP-Apt bioconjugates
cancer cells generally have made use of anti-
also presented remarkable effi cacy and reduced
bodies to deliver cytotoxic packages (Wu and side effects in vivo. These observations strongly
Senter, 2005). However, aptamers developed indicate a potential therapeutic application of
against PSMA as protein or another molecule of aptamer-nanoparticle bioconjugates to specifi -
biological importance potent target binders can cally target and destroy cancer cells.
be used instead of antibodies to deliver cytotoxic
Another approach with possible therapeutic
agents to cancer cells (Farokhzad et al. 2006). applications makes use of packaging RNA (pRNA)
Gelonin (Gel) is a protein toxin with n-glycosidase as part of the DNA-packaging machinery of the
activity promoting cell death by cleavage of a bacteriophage Phi29. This pRNA was genetically
specific glycosidic bond of rRNA thereby engineered to originate chimeric RNA that forms
promoting inhibition of protein synthesis and dimers via interlocking right- and left-handed loops
resulting in elimination of target cells. However, (Guo et al. 2005). Fusing pRNA with either
gelonin does not contain a translocation domain receptor-binding RNA aptamers, folate, small
such as those of many other ribosomal toxins do, interfering RNA (siRNA), ribozyme, or another
and is not incorporated into cells at considerable chemical group did not disturb dimer formation or
quantities (Rosenblum et al. 1999). In order to interfere with the function of the inserted moieties.
further improve its cytotoxicity, a recombinant Incubation of cancer cells with the pRNA dimer
Gel (rGel) was developed. Although this recom-
with one subunit harboring the receptor-binding
binant variant provoked some toxicity on target moiety and the other containining the gene
cells, membrane-permeability was not signifi -
expression-silencing molecule resulted in target-
cantly improved (Rosenblum et al. 1999). This cell recognition, uptake into these cells and subse-
problem was solved by chemical conjugation of quent silencing of anti-apoptotic gene expression.
rGel or its genetic fusion with the delivery The chimeric pRNA complex was found to be
package’s recognition molecules and by addition processed into functional double-stranded siRNA
of cysteine residues to form antibody immuno-
by the RNA-specifi c endonuclease Dicer. Animal
conjugates (Rosenblum et al. 2003; Better et al. trials confi rmed the suppression of tumorigenicity
1994). For instance, the cytokine VEGF was of cancer cells by ex vivo delivery (Guo et al. 2005;
coupled to gelonin and the resulting conjugate Khaled et al. 2005). These small-size RNA
specifi cally killed cancer cells overexpressing the nanoparticles will allow repeated long-term
VEGF receptor FLT-1 (Veenendaal et al. 2002). administration and avoid the problems of short
A selective drug that can home in a specifi c target retention time of small molecules and will also
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Ulrich et al
avoid the delivery problems of particles larger than protein and NA release time-spans would allow more
100 nm.
effi cient addressing of drugs to specifi c target tissues
Farokhzad and collaborators (2004) synthesized (Carrasquillo et al. 1999; Carrasquillo et al. 2001a;
a polylactic acid (PLA)-block-polyethylene Carrasquillo et al. 2001b).
glycol (PEG) copolymer with a carboxilic
Frazza and Schmitt (1971) developed a poly
terminal functional group (PLA-PEG-COOH), (lactic-co-glycolic) acid (PLGA) polymer, which
and encapsulated rhodamine-labeled dextran since then has been widely used in clinical proce-
inside PLA-PEG-COOH nanoparticles. These dures as suture for tissue engineering (Hasirci et al.
nanoparticles have negatively charged carboxilic 2001; Ma and Choi, 2001). PLGA may be locally
groups on their surfaces, which minimize unspe-
applied, allowing intralesional administration of
cifi c interactions with negatively charged NAs drugs while minimizing adverse systemic effects.
and, for instance, can be conjugated with amino-
The possibility of local application of PLGA consti-
modifi ed NAs. Clinical trials revealed that the tutes an important pharmacological advantage
presence of anti-PSMA aptamers at target cells is (Mallery et al. 2000; Moritera et al. 1991). The
increased 77-fold when it is bioconjugated to a employment of PLGA as a drug delivery system
nanoparticle (Farokhzad et al. 2004). The incuba-
for the therapy of ocular diseases did not reveal any
tion of protein-free nanoscale particles containing signs of ocular toxicity or infl ammatory processes
a receptor-binding aptamer or other ligands may even during long treatment periods (Moritera et al.
result in the binding and internalization of the 1991; Giordano et al. 1995). However, adverse side
trivalent therapeutic particles (Dtxl-NP-Apt or effects resulted from the sclerotomy as result of the
PLA-PEG-COOH-Apt) subsequently modulating delivery procedure of small encapsulated synthetic
prostate cancer cell apoptosis. These bioconju-
drugs. Carrasquillo et al. (2003) developed a drug
gates were based on materials which had been delivery system that continuously releases the
priorly approved for clinical use by the FDA. EYE001 anti-VEGF aptamer (Macugen) in a
Since these molecules are small, relatively stable, controlled fashion during signifi cant time spans
non-immunogenic and easy to synthesize, the when it is locally administered at the external area
translation of these bioconjugates into clinical of the sclera. The use of the proposed delivery
practice is facilitated. Therefore, therapeutic and system for the release of Macugen illustrates a
diagnostic nanoparticle-aptamer bioconjugates promissing alternative for the tansscleral delivery
will be shortly developed for other important of drugs for the treatment of ocular and choroidal
human diseases. In this regard, RNA molecules illnesses.
might be used as building blocks in many asso-
Another way to successfully home aptamers at
ciations in nanotechnology.
their subcellular targets is to express them in the
The delivery of macromolecules (including glob-
cells of interest. In this case the expressed drugs
ular proteins and aptamers) to the sclera as therapeutics would be useful for both the treatment of heredi-
in eye disease has been described in recent studies tary diseases as well as of viral diseases such as
(Ambati et al. 2000a; Ambati et al. 2000b). The trans-
AIDS. The TAR region of the RNA genome of
port and potential diffusion of molecules to the sclera HIV-1 is an attractive target for inhibitory NA-
takes place through an extensive surface area based drugs. TAR is a 57 nucleotide regulatory
containing a high percentage of water. Water is the element present at the 5′ end of every viral RNA
main constituent of the extracellular matrix containing particle. It exerts a crucial role on viral transcrip-
few cells and unchanging permeability during aging tion, as it is recognized by the ternary complex
(Olsen et al. 1998; Olsen et al. 1995; Boubriak et al. composed of the Tat viral protein (trans-activator
2000). The use of this route of administration could of transcription) and of two cellular proteins
avoid problems and limitations of other delivery named cyclin T1 and CDK9 (Herrmann and
approaches in the treatment of viral and systemic Mancini, 2001; Richter et al. 2002).CDK9 when
diseases (Kamei et al. 1999; Dayle, 2001; Lang, 1995). associated to Tat, hyperphosphorilates the
Since some transscleral delivery systems may be carboxy-terminal of RNA polymerase II and
destructive (i.e. iontophoresis) occasionally provoking subsequently activates the transcription machinery
retinal necrosis and gliosis (Lam et al. 1991), the ideal triggering the effi cient synthesis of the entire viral
approach would be to develop biodegradable polimeric RNA. A NA-based drug strongly interacting
particles with prolonged delivery capacity. Lenghtened with TAR competes with the formation of the
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Delivery systems of nucleic acids drugs
transcription complex and consequently inhibits decreased plasma levels of apoB protein, and
the trans-activation of the transcription apparatus. reduced total cholesterol concentration in mice
Thus, HIV-1 replication is compromised. Moreover, (Soutschek et al. 2004). Judge et al (2005) made
since the TAR element is located in close prox-
use of siRNA duplexes formulated in stable NA
imity to the 5′ end of the mature mRNA, a TAR lipid particles (SNALPs) to attain gene silencing
binder could interfere with the ribosomal machinery of apoB in mice (Zimmermann et al. 2006). More-
as well.
over, the general applicability of SNALP formula-
NAs, including antisense oligonuvleotides, tions for hepatic delivery of siRNA has been
siRNA and aptamers were employed in many demonstrated in animal models of HBV and Ebola
studies for targeting the TAR element (Turner et al. virus infection (Morrissey et al. 2005; Geisbert
2005; Yoshinari et al. 2004; Ducongé and Toulmé, et al. 2006).
1999). The anti-TAR RNA aptamer (Ducongé and
In oncology, direct delivery of siRNAs and viral
Toulmé, 1999) was optimized by chemical modi-
delivery of small hairpin (sh)-RNAs to tumors have
fi cations towards improved stability regarding been shown to successfully inhibit xenograft
nuclease resistance and decreased trans-activation growth in mouse models.
of transcription in cell nuclei extract assays
(Darfeuille et al. 2002a; Darfeuille et al. 2002b;
Darfeuille et al. 2004; Kolb et al. 2005; Toulmé Combination of siRNA and aptamers
et al. 2001). Other aptamers were also developed in therapeutics
interfering with HIV-1 gene expression, such as Technologies that mediate targeted delivery of
the anti-HIV Rev-binding aptamer (RBEapt) siRNAs are needed to improve their therapeutic
(Konopka et al. 1998). Konopka and collaborators effi cacy and safety for use in humans. Lupold
used cationic liposomes as delivery vessels et al. (2002) identifi ed two aptamers that bind
carrying the association of RBEapt and a ribozyme with low nanomolar affinity to the extracel-
that acts against the HIV-1 env gene inhibiting viral lular portion of PSMA. The two aptamers,
production (Konopka et al. 1998). These data denominated as xPSM-A9 and xPSM-A10, did
provide strong evidence for the therapeutical not share any consensus sequences and bound to
potential of NA ligands as anti-HIV agents when different sites of PSMA. Distinct modes of inhi-
their intracellular delivery is effi cient.
bition suggested that each aptamer identifi es a
unique extracellular epitope of recombinant
Delivery systems for siRNA
PSMA (xPSM). These aptamers were the fi rst
Conjugation of NA terminals with lipophilic recognizing specifi c prostate cancer markers. Chu
molecules has been reported to improve or direct et al. (2006) coupled siRNAs interfering with
cellular uptake. For example, siRNAs conjugated laminin and GAPDH gene expression by a
with cholesterol improved in vitro and in vivo modular streptavidin bridge to the aptamer A9
permeation of liver cells (Lorenz et al. 2004). A which binds to prostate-cancer cells. Comparison
number of approaches—including lipid-based of oligofectamine (Invitrogen) and aptamer-medi-
formulation, TransMessenger (Niu et al. 2006) and ated siRNA transfection resulted in similar inhibi-
complexation with polyethylenimine (Grzelinski tion of target-protein expression as evaluated by
et al. 2006), cholesterol-oligoarginine (Kim et al. real-time PCR experiments. PSMA endocytosis
2006), a protamine-Fab fusion protein (Song et al. in LNCaP cells is thought to predominantly
2005) and atelocollagen (Takei et al. 2004; proceed via clathrin-coated pits. The rate of inter-
Minakuchi et al. 2004)—have been shown to nalization has been previously measured using
facilitate delivery into tumor cells. Aptamer-siRNA antibodies directed against PSMA and also in the
chimeric RNAs have also been successfully used presence of the anti-PSMA aptamer A10. Inter-
to facilitate siRNA delivery in vivo (McNamara et al. nalization was shown to take place for both types
2006; Chu et al. 2006) (Fig.1 C).
of targeting agents within hours of binding
In 2004, Soutschek and coworkers demon-
(Farokhzad et al. 2004). Inhibition of target-gene
strated effective silencing of gene expression of expression was detected after 72 h of siRNA-
apolipoprotein apoB by intravenous administration aptamer transfection.
of chemically modifi ed siRNA which resulted in
McNamara et al. (2006) generated a chimera of
silencing of the apoB mRNA in liver and jejunum, A10 and a siRNA construct targeting polo-like
Drug Target Insights 2007:2
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Ulrich et al
kinase 1 (PLK1) and BCL2 (Yano et al. 2004;
Current activities of the CytRx Corporation are
Reagan-Shaw and Ahmad, 2005) as two survival concentrated in the development of small molecule
genes overexpressed in most human tumors (Takai drugs, RNAi drug discovery and DNA vaccines
et al. 2004; Eckerdt et al. 2005; Cory and Adams, and a delivery technology with multiple applica-
2005). Since Dicer also acts on chimeric RNAs, tions in the area of DNA vaccine and gene therapy.
aptamer-siRNAs are directed into the RNAi Its proprietary poloxamer compound, TranzFect,
pathway and silence their cognate mRNAs. has revealed good results regarding its transfection
Aptamer-siRNA chimera–mediated gene silencing ability, immunoadjuvant activity and toxicity
is dependent on Dicer activation and occurs via the profi le as prerequisites for DNA-based vaccines.
RNAi pathway. However, inhibition of Dicer acti-
This company also participates in the development
vation had no effect on transfected PLK1 siRNA-
of gene-silencing technologies for treatment of
mediated silencing, as 21- to 23-nucleotide Amyotrophic lateral sclerosis, type II diabetes,
containing siRNAs have been shown to bypass the CMV retinitis, obesity, and cancer. Targeting of
Dicer step (Murchison et al. 2005).
the epidermal growth factor (EGF) receptor by
The chimera A10-siRNA specifi cally bound to pegylated immunoliposomes carrying a plasmid
PSMA on the surface of LNCaP cells but did not coding for the shRNA prolonged the survival of
interact with PC-3 prostate cancer cells which do mice with intracranial human brain cancer (Zhang
not express PSMA (see scheme in Fig.1C). The et al. 2004). The observation that encapsulated
combination of shRNA and aptamer for specifi c oligonucleotides readily passed the blood-brain
gene expression control in target cells was accom-
barrier, encourages the development of NA-based
plished by An et al (2006). This research group therapies for neurodegenerative diseases (reviewed
constructed a vector containing the theophylline-
by Sa, 2004).
binding aptamer (Jenison et al. 1994; Zimmermann
et al. 1997) and the loop region of the shRNA Conclusions
targeting the enhanced green fl uorescent protein
(EGFP) under U6 promoter expression. When this In the nanotechnology fi eld, aptamers have the
construct was co-transfected with a construct potential to act as targeting molecules by directing
coding for EGFP expression (pEGFP-N1) into the delivery of nanoparticles to antigens present on
HEK cells, shRNA-induced EGFP gene expression the surface of target cells. In general terms, thera-
silencing was dose-dependently inhibited by peutic nanoparticles are components of specialized
increasing concentrations of theophylline, as delivery vehicles of an encapsulated drug. Drug
aptamer-bound theophylline interfered with the release should occur in a regulated and defi ned
Dicer-cleavage site. This study proved the feasi-
manner. Depending on the therapeutic demand,
bility of modulation of gene expression under the such devices are designed to ensure continuous or
control of intracellular proteins or cell metabolites immediate drug release. The combination of
(An et al. 2006).
targeted delivery and controlled release of drugs at
affected tissue sites will lead to the development of
“smart therapeutics”, which are more effective and
Recent Clinical Developments
will have less undesired side effects than drugs
One of the fastest approvals ever obtained for a available today. Several studies have shown that
drug by the FDA was for imatinib (imatinib nanoparticles can be attached to NAs in a way that
mesylate [Gleevec], from Novartis) for the treat-
NA-binding properties to their targets are preserved,
ment of chronic myelogenous leukemia (CML). thus reducing their potentially associated delete-
Although the incidence of CML is low, the rate of rious side effects. These new approaches can
cure with conventional treatment is poor. Gleevec supplement the conventional chemotherapy and
is a tyrosine kinase inhibitor and affects only the radiotherapy in cancer treatment, prevent drug
leukemic cells that are caused by fusion of two resistance and damage to normal tissues. In view
genes, bcr and abl, a chromosomal shuffling of the impact of the genomic revolution on
between chromosomes 9 and 22. Although expen-
improving medicines and healthcare, state-of-the-
sive, Gleevec, an oral drug, led to remission in 90% art NA carriers together with highly specifi c oligo-
to 95% of CML-relapsed patients. However, it has nucleotide drugs may help in reducing the side
not worked well in blast crisis.
effects of drugs during therapy.
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