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Advanced Drug Delivery Reviews 64 (2012) 1508–1521
Contents lists available at SciVerse ScienceDirect
Advanced Drug Delivery Reviews
journal homepage: www.elsevier.com/locate/addr
RNAi-based nanomedicines for targeted personalized therapy☆
Ala Daka, Dan Peer ⁎
Laboratory of Nanomedicine, Department of Cell Research and Immunology, George S. Wise Faculty of Life Science, Israel
Center for Nanoscience and Nanotechnology, Tel Aviv University, Tel Aviv, 69978, Israel
a r t i c l e
i n f o
Article history:
Received 5 April 2012
Accepted 13 August 2012
Available online 5 September 2012
Keywords:
Short interfering RNA (siRNA)
In vivo
Systemic delivery
Nanoparticles
Clinical trials
Personalized medicine
a b s t r a c t
RNA interference (RNAi) has just made it through the pipeline to clinical trials. However, in order for RNAi to
serve as an ideal personalized therapeutics and be clinically approved—safe, specific, and potent strategies
must be devised for efficient delivery of RNAi payloads to specific cell types, which despite the immense
potential, remains a challenge. Through evaluating the recent reported studies in this field, we introduce
the progress in designing targeted nano-scaled strategies that are anticipated to overcome the delivery drawbacks and along with the exciting “omics” discipline to personalize RNAi-based therapeutics.
© 2012 Elsevier B.V. All rights reserved.
Contents
1.
2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
RNAi: A powerful approach for personalized medicine . . . . . . . . . . . . .
2.1.
RNAi: An advantageous strategy over conventional therapeutics . . . . .
2.2.
Personalizing RNAi-based medicine . . . . . . . . . . . . . . . . . .
2.2.1.
RNAi-based screening
. . . . . . . . . . . . . . . . . . . .
2.2.2.
The design of specific siRNAs . . . . . . . . . . . . . . . . .
Strategies for in vivo delivery of RNAi molecules . . . . . . . . . . . . . . . .
3.1.
Local delivery of RNAi molecules . . . . . . . . . . . . . . . . . . . .
3.2.
Systemic delivery of RNAi molecules . . . . . . . . . . . . . . . . . .
3.2.1.
Delivery challenges: Rapid clearance of RNAi molecules . . . . .
3.2.2.
Delivery challenges: Stability of the RNAi molecules . . . . . .
3.2.3.
Off-target effects of induced RNAi: Immunostimulatory effects .
3.2.4.
Off-target effects of induced RNAi: miRNA-like effects . . . . .
3.2.5.
Off-target effects of induced RNAi: Saturation of the RNAi machinery
Systemic NP-mediated delivery of RNAi molecules . . . . . . . . . . . . . . .
4.1.
Passive delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.1.
Lipid-based nanoparticles (LNPs) . . . . . . . . . . . . . . .
4.1.2.
Cationic LNPs . . . . . . . . . . . . . . . . . . . . . . . .
4.1.3.
Lipidoids . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.4.
Polyplexes . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abbreviations: mAb, monoclonal antibody; ApoB, apolipoprotein B; BD, biodistribution; CME, clathrin-mediated endocytosis; CNT, carbon-nanotubes; DC-6-14, O,O′ditetradecanoyl-N-(α-trimethylammonioacetyl) diethanolamine chloride; DOPE, dioleoylphosphatidylethanolamine; DOTAP, N-[1-(2,3-dioleoyloxy)-propyl]-N,N,N-trimethylammonium
methylsulfate; DOTMA, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride; dsRNA, double stranded ribonucleic acid; EPR, enhanced permeability and retention; I.V.,
intravenous; miRNA, micro ribonucleic acid; MPS, mononuclear phagocytic system; MW, molecular weight; NP, nanoparticle; nt, nucleotide; PEG, polyethylenglycol; PEI, Polyethyleneimine;
PC, phosphatidylcholine; P.I., post injection; PK, pharmacokinetics; RES, reticuloendothelial system; RISC, RNA-induced silencing complex; RNA, ribonucleic acid; shRNA, short hairpin
ribonucleic acid; SNALP, stable lipid-nucleic acid particle; T1/2, half time; TLR, Toll-like receptor.
☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Personalized nanomedicine.”
⁎ Corresponding author at: Center for Nanoscience and Nanotechnology, Tel Aviv University, Tel Aviv, 69978, Israel. Tel.: +972 3640 7925; fax: +972 3640 5926.
E-mail address: [email protected] (D. Peer).
0169-409X/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.addr.2012.08.014
Author's personal copy
A. Daka, D. Peer / Advanced Drug Delivery Reviews 64 (2012) 1508–1521
4.1.5.
Carbon-nanotubes (CNTS) . . . . .
Active, target-specific RNAi delivery . . . . .
4.2.1.
Peptide-based targeting moieties . .
4.2.2.
Aptamer/siRNA chimeras . . . . . .
4.2.3.
Other small molecules-siRNA NPs . .
5.
Intracellular delivery of NP-associated RNAi molecules
5.1.
Delivery to the cell membrane . . . . . . .
5.2.
Endosomal escape . . . . . . . . . . . . .
6.
NP-mediated RNAi: Clinical studies . . . . . . . . .
7.
Conclusions . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . .
4.2.
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1. Introduction
RNAi is a specific regulatory mechanism of most eukaryotic cells, in
which small double-stranded ribonucleic acids (dsRNAs) efficiently control gene expression in a complementarity-dependent manner. This natural mechanism, which involves diverse families of small non-coding
RNA regulators, is presumed to protect against pathogenic infections as
well as regulate various biological pathways [1]. Among the endogenous
regulators, microRNAs are of great importance in controlling numerous
physiological processes. A long hairpin-structured dsRNA (pre-miRNA)
is transported out of the nucleus to the cytoplasm, where it is cleaved
by RNase III endonuclease DICER to generate a mature miRNA. The
double-stranded miRNA, which is 21–25 nt long with 5′-phosphorylated
ends and 2-nt 3′ overhangs, incorporates into a ribonucleoprotein complex, the RNA-induced silencing complex (RISC) that unwinds the RNA
duplex and discards the sense strand. The antisense strand (guide
strand) then guides RISC to its target mRNA. The miRNA guide strand
most often pairs partially with the mRNA transcript and triggers its
translation repression or leads to its degradation [2,3].
Utilizing this native mechanism for therapeutic and diagnostic purposes can be achieved by exogenous delivery of synthetic RNAi molecules.
However, due to their net negative charge and relatively large size compared to vasculature effective pore size, RNAi molecules are less likely to
readily cross biological barriers; thus, efficient systemic delivery necessitates the use of physical protection (i.e. carriers/reagents) and chemical
modifications that combined protect RNAi molecules until reaching
their site of action and facilitate cell penetration yet maintaining their
functionality.
One approach to exogenously trigger RNAi in cells is the use of
short-hairpin RNA (shRNA) or miRNA mimics. Most commonly, these
strategies are applied by introducing DNA [4] or stem-and-loop
containing RNAs into the nucleus in a viral vector-based manner where
shRNAs are synthesized and processed via the miRNA pathway [5].
Although shRNA method is considered to be an efficient way of inducing
RNAi, mainly because of the induction of prolonged and stable
gene-silencing that is favored in the case of chronic diseases [6,7], the
toxic effects that have been reported in animals models and patients
treated with shRNAs [5,8–10] limit its clinical use. These effects are
apparently due to: 1) competition with and overload of endogenous
RNAi machinery. 2) Imperfect complementarity of shRNA with its target
mRNA that may lead to nonspecific silencing and off-target effects. 3)
Stimulation of the immune system. 4) Insertional mutagenesisinduced tumorigenicity possibly enhanced upon viral delivery, and, 5)
undesirable long-term expression especially in non-chronic disease
models [10]. Furthermore, shRNA-based RNAi induction necessitates
intranucleus localization; an additional hurdle that must be mitigated.
Consequently, non-viral approaches provide an alternative way to
induce gene silencing. Conjugating or incorporating RNAi molecules to
or into nanoparticles (NPs), are the major non-viral nano-scaled delivery vehicles that have been recently developed, and been approved to
overcome major viral vector drawbacks. Nanobiotechnology offers a
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vast number of nanomaterials enabling the development of a variety
of novel systems (such as liposomes, nano/micro-spheres, nanotubes
etc.) feasible for biological and medical applications [11,12].
In this review, we present the major impediments for translating
RNAi from a genomic tool to clinical practice, review the recent progress
in designing targeted nano-scale delivery systems to tackle these impediments and highlight the necessity of “omics” technology that
anticipates revolutionizing and expediting the development of the
area of personalized diagnostics, disease management and therapeutics.
2. RNAi: A powerful approach for personalized medicine
Introducing RNAi in medicine is achieved by designing specific RNAi
regulators such as miRNA mimics and small interfering RNAs (siRNAs).
Antagomirs are novel synthetic 21–23 nt single stranded miRNA analogs
that have been recently developed for specific miRNA silencing. Since
miRNAs are involved in many biological and pathological processes,
and given that Antagomir-mediated gene-regulation is differential and
selective as it induces silencing of specific miRNA targets derived from
the same transcript [13], it is therefore rational to utilize it for therapeutic
intervention. However, because most of the RNAi-related studies and
NPs developing were conducted with siRNAs rather than miRNA-based
molecules, the focus of this review is mainly on siRNA-carrying systems.
siRNA is a chemically synthesized RNA-duplex, which is 19–23 nt in
length. siRNAs are designed so they have 2-nt 3′ overhang, similar to
endogenous miRNAs, allowing DICER to recognize and further process
them. However, while miRNA targets partially complements mRNA,
siRNAs trigger a sequence-specific mRNA cleavage of perfectly complementary targets [14,15]. This feature of specific gene-regulation combined with the high potency and safety compared to conventional
medicines or other classical anti-sense and gene silencing methods
(such as antisense oligonucleotides and shRNAs [8,16]) make it possible
to utilize siRNA as a safer platform for therapy in personalized medicine.
2.1. RNAi: An advantageous strategy over conventional therapeutics
RNAi-induced gene silencing mirrors the inhibitory effects of conventional pharmaceuticals, such as protein-based drugs (e.g. antibodies and
vaccines) and small molecules. The inhibitory effects of these drugs is
mainly achieved by blocking their targets function, however some
disease-related molecules, primarily proteins, do not have enzymatic
function or have a conformation that is not accessible to conventional
drugs or small molecules compounds, hence are considered as
“non-druggable” targets. Comparable "non-druggable" molecules have
been successfully targeted by RNAi approach in vivo [17–21] demonstrating an exclusively allele-specific gene silencing [22,23]. Additionally,
blocking target's gene expression rather than its activity is more efficient,
as single mRNA molecule translates multiple copies of protein. Another
advantage of RNAi over conventional pharmaceuticals is the ease of synthesis and production of potent regulators. Opposing to protein-based
drugs, RNAi synthesis does not necessitate cellular expression systems
Author's personal copy
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or refolding steps. As for small molecules-based drugs, the major disadvantage is the slow and difficult lead identification and optimization
[24], whereas the identification of functional and specific RNAi constructs
is rapid and involves well-demonstrated strategies and algorithms that
facilitate their optimization.
However, whilst protein and some small molecules-based drugs do
not require intracellular access to obtain functionality, RNAi-based therapeutics necessitate intracellular delivery, an additional challenging
task for the development of a new efficient RNAi-based therapeutic.
2.2. Personalizing RNAi-based medicine
Given that variation in gene expression patterns may lead to variation
in response to a treatment often observed among different patients, personalized medicine aims to use specific therapeutics best suited for an individual considering pharmacogenetic and pharmacogenomic data [25].
The arising “omics” technology field, which beyond genomics, proteomics
and metabolomics that contribute to further understanding of the bases
of diseases as well as the interaction of the transcriptomes with their
environment, leading to identify disease-related genes and molecular
determinants; it also refers to toxicogenomics and pharmacogenomics,
which provide data on reciprocal interactions of the RNAi-based medicine
and the genetic and physiological processes, facilitating the design of
novel individualized RNAi constructs accordingly [26].
2.2.1. RNAi-based screening
Genome-scale RNAi analysis is one of the more powerful genomics
strategies nowadays. It was first conducted in model organisms
(Caenorhabditis elegans and Drosophila). Recently, number of studies
utilizing RNAi-based screens was reported in human cells, for example,
it was used to identify a number of human host genes affecting influenza A virus replication [27] and west Nile virus infection [28]. In another
report, siRNAs were utilized for whole genome lethality study;
Tiedemann et al. transfected myeloma human cell lines with siRNA constructs against potential “druggable” targets of myeloma and measured
viability. Survival genes were determined as genes that induced cell lethality upon siRNA-related silencing. This application allows identification of important survival genes for the non-curative myeloma cancer
[29]. A significant advance in this field was described by Neumann et
al. who developed an automated system for siRNA screening by
time-lapse fluorescent imaging in live human cells for investigating
the chromosome segregation during cell division. This automated platform allows detailed time-resolved and quantitative analysis of phenotype signatures and makes genome-scale RNAi screening applicable in
human live cells, overcoming the limitations of end-point RNAi-based
screens [30].
2.2.2. The design of specific siRNAs
Depending upon the comprehensive data on novel surface and
intracellular determinants and cellular pathways involved in various
pathological processes identified by pharmacogenomic and epigenomic
analyses, it is now possible to design specific siRNAs. The synthesis of
personalized siRNA constructs requires well-established designing
and bioinformatic tools that identify a 21–23 nt with 3′ overhang
double-stranded siRNA sequence against single target from siRNA
libraries. This is followed by small-scale synthesis of siRNAs panel and
conducting in vitro assays to determine specificity and efficacy and to
eliminate off-target effects and cytotoxicity. Based on the in vitro studies, chemical modifications and refinements of the siRNA construct are
applied while preserving or improving its potency, specificity and
stability [31,32].
Owing to the differences in RNAi-related responses, off-target
events and immune response between in vitro and in vivo systems, it
is reasonable to identify numbers of siRNA sequences for in vivo
applications, that commonly are the most specific, potent and safe
lead constructs. Due to the availability of bioinformatic algorithms
enabling the design of single siRNA construct that is cross reactive in
rodents and primates, the availability of transgenic and knockdown rodents and the assumption that the physiology of non-human primates
is comparable with that of humans, in vivo safety screening conducted
in rodents and non-human primates presupposed to be of great importance though not sufficient when progressing to clinical studies [33].
3. Strategies for in vivo delivery of RNAi molecules
The main aim of RNAi applied in vivo is to achieve safe gene-specific
silencing via tissue-specific targeting using efficient amounts of RNAi
molecules. In vivo delivery of siRNA could be accomplished via local or
systemic administration routes, depending on the tissues/organs that
are targeted.
3.1. Local delivery of RNAi molecules
Various approaches for local delivery of siRNA molecules have been
recently applied in animal models. This strategy is based on injection of
naked or formulated siRNA in vivo in a localized manner, and mainly
includes: intranasal [34–36], intraocular [37,38], intratumoral [39,40],
intramuscular [41], and intracerebral [42,43] approaches.
Local administration is site-specific, i.e. the injected molecules do
not circulate through the body, which offers high bioavailability of
the RNAi molecules at the target tissue with low effective dosage,
and allows utilizing simple siRNA formulations or even naked molecules. As exemplified in a study by Niu et al., effective gene silencing
and tumor suppression upon direct subcutaneous and intraperitoneal
injection of siRNA complexed with commercial transfection reagent
showed specific gene silencing of human papillomavirus (HBV) 16
E6 oncogene in a cervical cancer model [44]. However, despite the
technical ease of the delivery approach, efficiency and selectivity,
local administration is applicable for restricted types of tissues,
which are technically accessible for invasive delivery.
3.2. Systemic delivery of RNAi molecules
In contrast to mucosal and subcutaneous tissues, most of body organs
and cells are not accessible to RNAi molecules upon direct administration; accordingly systemic delivery strategies must be devised. The fact
that nucleic acids in general are exposed to phagocytosis, nuclease degradation and are membrane-impermeable makes systemic RNAi delivery
more complicated and challenging. Due to these hurdles as well as anatomical barriers, RNAi delivery entails significant optimizations prior to
administration [45]. The first barrier the delivered RNAi molecule has
to overcome before reaching its site of action within the target cell cytoplasm is the circulatory system, where it is imposed to deal with serum
proteins including nucleases, kidney filtration and the immune system.
Once the RNAi molecule reaches the target cell, further obstacles
concerning endocytosis routes and endogenous RNAi pathways have to
be tackled. Combined together, those impediments are known as gene
silencing off-target effects, i.e. undesirable effects that could alter the
siRNA-induced phenotypes and interfere with analyzing the obtained
results. Below we introduce the major barriers that must be overcome
when RNAi molecules are systemically administered while preserving
their function.
3.2.1. Delivery challenges: Rapid clearance of RNAi molecules
Naked, unmodified RNAi molecule, faces a number of obstacles
during its circulation in the blood stream; the first one is its relatively
small size compared to the glomerular effective pore size - as molecules less than 50 kDa (~ 10 nm) are exposed to glomerular filtration
in the kidney and excretion into the urine [46,47]. Studies show that
systemically administered naked siRNAs preferentially accumulated
in the kidney (40 times higher than other organs) and excreted into
urine within one hour [48,49]. Even though accumulation of siRNAs
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in the kidney may be favorable for selective renal targeting and gene
silencing [48], recognition of naked siRNAs by the phagocytic system
is associated with immunological and cytotoxic stimulation, that
could occur right after injection through activation of circulating
mononuclear phagocytosis or inside the cytoplasm and endosome
compartments of the target cell.
3.2.2. Delivery challenges: Stability of the RNAi molecules
Although dsRNAs are more stable compared to other oligonucleotides (ssRNA or DNA), unmodified siRNAs are still exposed to nuclease
activity when administered systemically, however, chemical modifications of the delivered siRNA could overcome this degradation. Several
researchers have studied the effect of modifications on siRNA function
and stability [24,50]. For note, increasing stability of the modified
siRNA should be accompanied with tolerance to the RNAi machinery;
furthermore, more effective modifications possess the ability to increase
silencing activity as well as biodistribution (BD) and pharmacokinetic
(PK) properties.
Since 2′-OH group of the ribose is not essential for siRNA to enter the
RNAi machinery [51], diverse modifications at the 2′-position in both
strands are widely used, such modifications are commonly known as
2′-modifications, and mainly referred to 2′-O, 2′-Methyl-nucleoside
(2′-O-Me) and 2′-deoxyflouridine (2′-F) modifications. Another modification of the 2′ position of the ribose is the use of locked nucleic
acids (LNAs), in which a methylene linkage is generated between the
2′ and 4′ positions [52].
An additional type of chemical modification is the phosphorothioate
(P= S) linkage at the 3′ end. PS linkage is generated by replacing one of
the nonbridging oxygen atoms on the phosphate between two riboses
with a sulfur atom, resulting in exonuclease resistance [24].
These siRNA chemical alterations have been shown to successfully
enhance siRNA stability both in vitro and in vivo, and in some cases to
enhance potency, reduce interference with endogenous RNAi pathways and to increase serum and thermal stability [51–55], yet, more
research in this field is needed as it has been reported that bulkier
modification in 2′-position could interfere with RNAi machinery [56].
3.2.3. Off-target effects of induced RNAi: Immunostimulatory effects
dsRNA induces immune response as a defense mechanism against
viral infection [1] upon interaction with RNA-binding proteins such as
Toll-like Receptors (TLRs) and protein kinase receptor (PKR). This
recognition occurs on the cell surface or within the cell (in the cytoplasm or endosome compartments) and leads to innate immunity
activation, i.e. production of pro-inflammatory cytokines, such as IL-6
and TNFα, and triggering type I Interferon (IFN) pathway [57]. siRNAs
can stimulate the immune system in a sequence-independent manner
as in the induction of TLR3 [58] and IFN pathways [59], as well as in a
sequence-dependent way as in the case of uridine (U) or guanosine
adenine/uridine (GA/GU)‐rich regions recognition via TLR7 and TLR8
[60–62].
Immunostimulatory effects can also be length-dependent. While
short siRNAs (12 and 16 nt long) have been reported to poorly stimulate immune cells, longer constructs increased significantly cytokine
production—independent of their sequence [61]. It is important to
note that immunostimulation by siRNA is cell type-dependent
[60,63], for example, though non-immune cells do not express TLRs
or express them at low levels, they retain the ability to recognize exogenous RNAs via alternative pathways such as the PKR pathway [64].
Besides optimizing the sequence [65], structure [66,67] or length [61]
of the siRNA, chemical modifications can also be applied for mitigating immunogenicity [68].
Of note, immunogenic siRNA sequences could be deliberately
applicable in specific treatments such as viral infections or cancer
[64], where activation of the immune system is required in order to
ameliorate the disease.
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3.2.4. Off-target effects of induced RNAi: miRNA-like effects
Despite the fact that siRNA-directed mRNA cleavage and the
native miRNA-directed translational repression are distinct pathways
that have different requirements [69,70]; the two RNAi mechanisms
still share common components and entry points, such as the
DICER-RISC processing machinery. This suggests that transfected
siRNA constructs may function as or interfere with the endogenous
miRNAs as demonstrated below.
By applying Omics tools via microarray profiling and analyzing gene
expression of different transcripts (i.e. gene expression signatures)
Jackson et al. have examined different siRNA sequences that target the
same gene. The rationale for this tool is that different siRNA sequences
against the same gene would generate similar gene silencing signatures
only if siRNA-induced gene silencing was selective against the targeted
mRNA transcript. Their data showed distinct gene silencing signatures
that are siRNA sequence-dependent. Those different signatures belong
to on- and off-target mRNA transcripts, that is, specific (targeted) and
unintended gene silencing, respectively [71]. The off-target gene silencing occurred even when mRNA transcripts partially hybridized with the
siRNA, which suggests the existence of a common feature on those
mRNA transcripts inducing that non-specificity. Studies reported that
a core region in the 3′-untranslated region (3′-UTR) of such mRNA transcripts shares complementarity with residues in the 5′ of the antisense
strand of the siRNA duplex [71–73]. This core region on siRNA is comparable to the “seed region” on miRNAs, which has a key role in mRNA target recognition in the endogenous RNAi pathway [74,75]. Thus, when
exogenous siRNA enters and arranges in the RISC complex in such a
conformation similar to that of the native miRNA, an “miRNA-like
off-target gene silencing” occurs and such non-specific signatures
observed by Jackson et al. are obtained [76,77]. It is of great importance
to note that these gene silencing signatures are mRNA transcript
sequence-specific [78], i.e. the signatures of siRNAs against different
mRNA transcripts are not similar, indicating that the off-target effects
are not only siRNA sequence-dependent but also target-specific.
3.2.5. Off-target effects of induced RNAi: Saturation of the RNAi machinery
Cross-reaction with endogenous RNAi machinery side effects is most
likely to occur upon shRNA use rather than siRNA. As above-mentioned,
shRNAs are delivered to the nucleus and enter the miRNA pathway at
early stages; therefore all components of the miRNA machinery could
be saturated by using high doses of exogenous dsRNA, particularly in
the case of the sustained expression of shRNA. These cross reactionrelated side effects may inhibit the miRNA pathway and cause cell toxicity. In their study, Grimm et al. [79] revealed that treatment with different shRNA constructs given to mice, led to severe toxicity in 73% of
the cases and to 47% lethality due to liver failure. The shRNA-related
lethality observed was dose-dependent and not shRNA/target-specific;
moreover, it did not require the presence of shRNA target, which hints
at competition with and saturation of one or more component of the
endogenous miRNA pathway. Those side-effects were eliminated
upon lowering the shRNA levels.
Even though exogenous siRNA enters the RNAi pathway at a later
step compared to shRNA and directly incorporates into the RISC, an
evidence for miRNA pathway saturation by siRNA has been recently
arisen [34,80], although can be minimized by reducing the delivered
siRNA amounts [34,81]. On the other hand, mismatches between the
delivered siRNA and the target mRNA could cause gene silencing via
miRNA-related pathway, which leads to translational inhibition without altering mRNA levels bringing on competition with the native pathway [82]. Interestingly, it is not the number of mismatches between the
siRNA and mRNA sequences but the position of the mismatches,
suggesting that structure stability of the siRNA determines whether
the siRNA enter the miRNA pathway or not. This indicates that sequence
complementarity is a crucial factor though not sufficient.
Altogether, while some microarray profiling tests show that siRNA
molecules could target mRNA sequences in a specific manner [83], it
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is now well established that siRNA silencing entails nonspecific off-target
gene silencing [71,78]. These effects could be a concentrationdependent, as lowering the siRNA concentration minimizes these
nonspecific effects [34,84] albeit not eliminating them [71]. Better
approaches for mitigating nonspecific off-target silencing are: 1) using
more than one duplex (i.e. siRNA pool) against the on-target mRNA, in
such a way different sequences target distinct and distinguishable
regions in the targeted mRNA, which minimize miRNA-like off-target
effects as well as significantly reduce interference with endogenous
gene silencing mechanisms. 2) Avoiding complementarity between
non-targeted mRNAs, especially in the 3′-UTR and seed region [73]. 3)
The use of chemical modifications, for instance the use of 2′-O-methyl
modification on positions 1 and 2 in the sense strand of 8 different
siRNAs was shown to reduce off-target silencing both at the mRNA and
protein levels without affecting the on-target mRNA silencing [85].
Collectively, those data highlight the necessity of the development
of siRNA off-target prediction algorithms that may optimize the construct of the delivered siRNA. Moreover, a physical approach in overcoming in vivo instability of siRNAs and the other off-target effects is
by complexation of the siRNA with a carrier/vector. The design of
appropriate siRNA carriers facilitates its delivery and trafficking to
and within the cell, and makes it feasible for in vivo application, as
these strategies: 1) mask the siRNA molecules from the immune system; 2) exceed the size range for kidney filtration, thus minimize
rapid clearance [47]; 3) protect the siRNA molecules from nuclease
activity and; 4) facilitate endothelial wall and plasma membrane penetration. Also, targeted delivery strategies increase the local concentration of siRNA at the site of action thus mitigates nonspecific
biodistribution and adverse effects accompanied with systemic delivery [50,86].
4. Systemic NP-mediated delivery of RNAi molecules
Challenges for in vivo delivery of RNAi molecules could emerge at
any level and are dependent on the route of administration; although
local administration is favored over systemic delivery it is not always
manageable for clinical application. The requirements for effective and
safe delivery of naked RNAi molecules such as avoiding renal clearance,
stability in the circulatory system, lack of immunogenicity, biocompatibility, immune tolerance of modifications and functionalizing, the ability to pass through biological barriers and specific targeting seem to
hold true also in the case of carrier-formulated RNAi systems designed
for clinical use. Furthermore, the carrier itself should meet other criteria
[87], for instance it should: have low tendency of aggregation, which
could induce toxicity and interfere with effective delivery, be soluble
or colloidal in water, have an extended circulating half-life (t1/2) that
increases effectiveness, release its payload into the cytoplasm before
reaching the lysosome and have a long shelf life [86].
The developing field of nanotechnology offers a wide range of NPs
for delivery of RNAi regulators. By definition, for a device to be considered a nano-scaled compound it or its essential component(s)
must be man-made and in the range of 1–1000 nm, at least in one
dimension [11]. When selecting the most suitable tool for delivery,
one should take into account the route of administration as well as
properties and structural characteristics of the carrier such as size,
shape, charge and other surface characteristics of the NP, as these features affect pharmacokinetics (PK) and biodistribution (BD) profiles
of the carrier. Accordingly, well-structured and fine tunable design
of the NP would result in developing adequate and appropriate device
for RNAi delivery [88].
4.1. Passive delivery
Passive delivery takes advantage of highly selective anatomical
and functional features of the targeted tissue to specifically deliver
the desired therapeutics. A leading approach of passive delivery is
exploiting the enhanced permeability and retention (EPR) effect for
solid-tumor targeting. The enhanced and abnormal angiogenesis
along with the poor lymphatic drainage characterized with tumorigenicity cause extensive permeability of blood vessels and high retention of macromolecules in tumor tissue, respectively [89,90]. While
free and low molecular weight (MW) drugs diffuse freely in and out
tumor tissues, macromolecules (> 40 kDa) and NPs between 100
and 200 nm do not easily diffuse back to the blood stream, hence
accumulate in the tumor tissue [89,91–94]. EPR phenomenon has
also been reported in various human solid tumors as well as in
inflammatory tissues [95].
In addition, passive delivery can be influenced by the NP formulation, for instance the passive delivery of naked siRNA molecules and
NPs ≤10 nm to the kidney upon systemic administration. On the
other hand, particles larger than 100 nm, particularly lipid-based
NPs, preferentially accumulate in organs of the reticuloendothelial
system (RES) better known now as the mononuclear phagocytic system (MPS) [17,68,96–98] due to high blood flow and fenestrated vasculature in these tissues. Mononuclear phagocytes of the MPS located
in the liver (Kupffer cells), lung and spleen take up foreign particles
following their opsonization by serum proteins via several interactions. Thus, size, geometry, surface charge and hydrophobicity of the
NP - could influence recognition by and accumulation within cells of
the MPS. An increase in phagocytic activity is observed upon using
highly charged NPs, prominently cationic, as well as hydrophobic particles. In order to avoid opsonization, strategies that mask the NP
charge and hydrophobicity may be applied, such as PEGylation, a
widely used method for NPs stabilization, referred to insertion of
the biocompatible poly-ethylene-glycol (PEG) derivative polymers
to the surface of the NP providing natural charge and hydrophilicity,
thus minimizing aggregation and opsonization [99–101].
In the next subsections we evaluate NP systems that make use of
such physiological mechanisms for passive delivery. These data are
summarized in Table 1.
4.1.1. Lipid-based nanoparticles (LNPs)
Nano-scaled liposomes are lipid-based particles ~100 nm in diameter
that consist of lipid bilayer envelope and hydrophilic aqueous core. Neutral liposomes are characterized with low immunogenicity and toxicity,
biocompatibility and ease of preparation, thus they have been investigated extensively [102,103]. Widely used neutral liposomes contain various
combinations of phosphatidylcholine (PC, mainly 1,2-Oleoyl-sn-Glycero3-phosphatidylcholine—DOPC), phosphatidylethanolamine (PE, mainly
1,2-Dioleoyl-sn-Glycero-3-phosphatidylethanolamine (DOPE)) and cholesterol [102], while the later two lipids are helper lipids that facilitate
endosomal escape of the payload to the cytoplasm and increase stability
of the liposome, respectively [104,105]. 65% siRNA encapsulation into
DOPC liposomes has been achieved [106], along with significant reduction in protein expression, tumor growth and metastasis in ovarian cancer
and human melanoma models in mice [106]. This tumor-targeted delivery was attained due to EPR effect of tumor tissues.
4.1.2. Cationic LNPs
Cationic lipid-based NPs that interact with the negative RNAi molecules as well as with the cell membrane, thus stabilizing the NP and
enhancing cell uptake, are frequently used in cell-culture assays
[102]. However, due to high levels of opsonization by serum proteins,
immunogenicity, accumulation near the vasculature and uptake by
the liver and spleen, specialized systems such as SNALPs those neutralize the NPs net charge with a “stealth coating” such as PEG
[47,99–101,105–107], increase the NP circulation t1/2 and improve
its PK would be discussed here. On the other hand, other factors
beside the net charge should be considered when designing NP for
delivery, e.g. the hydrodynamic diameter; as even PEGylated NPs
>100 nm may exhibit higher protein adsorption and thus lower
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Table 1
RNAi-based NPs for systemic administration.
Delivery system
NP formulation
Targeting mechanism
Target tissue
References
Lipid-based NP (LNPs)/
lipoplexes
Cationic LNPs
Lipidoids
Polyplexes
DOPC liposomes
EPR
Ovarian tumor
[106]
SNALP
Epoxide-based lipidoids
JetPEI/LNA-siRNA polyplex
MPS
MPS
Aggregation-induced retention,
Mucoadhesion
Mucoadhesion
EPR
MPS
Antibody-directed delivery
Antibody-directed delivery
Antibody-directed delivery
Liver
Liver
Lung, Liver, Kidney
[17,98,108]
[97,109]
[50]
Kidney
Liver tumors, Inflammatory cells
Liver
Dendritic cells
Gut leukocytes, T lymphocytes and monocytes,
Melanoma tumors, activated leukocytes
[50]
[92,94]
[116]
[117]
[118,119]
[120,122]
Integrin-targeting peptide
AchR-targeting peptide
PSMA-targeted aptamer
σ receptors-targeted Anisamide
Tumor neovasculature
AchR-expressing neurons
Prostate cancer cells
Tumor cells
[114,122]
[123]
[124,125]
[126]
RBP mediated targeting
TfR-mediated targeting
NAG-mediated targeting
Chol-mediated targeting
Hepatic stellate (HS) cells
Tumor cells expressing TfR
Liver heptocytes
Liver and jejunum
[127]
[128]
[129]
[13,130]
Chitosan/LNA-siRNA polyplex
Atelocollagen
Carbon nanotubes (CNTs) To17-SWCNT
Immuno-liposomes
I-tsNP
Protein-based targeting
Antibody-protamine fusion
systems
protein
RGD NPs
RVG-9R NPs
Aptamer/NPs
RNA A10/siRNA NP
Targeted cationic lipid-based PEG–Anisamide–LDP
NPs
VitA-Lipotrust liposomes
CDP NPs
Tf-CDP/siRNA
DPCs
PEGylated NAG-PBAVE DPCs
Chol conjugates
Chol/Antagomer122, Chol/siRNA
circulating t1/2 and permeation into tissues as well as higher MPS
clearance [47].
SNALP is a 100–150 nm lipid-based particle that encapsulates
siRNA within its layers. It is composed of cationic and fusogenic lipids
that facilitate cellular uptake and endosomal escape. The lipid bilayer
of SNALP is coated with PEG polymer [68]. Tissue BD assays in rodents
show significant delivery of SNALPs to the liver. Several studies report
minor liver enzymes release upon SNALP application, otherwise the
NPs are well tolerated [17,68].
In the first RNAi-induced gene silencing studies reported with SNALP
and conducted in non-human primates, Zimmermann et al. reported significant (>90%), specific and durable dose-dependent gene silencing of
apolipoprotein B (ApoB) by single injection of SNALP-formulated siRNA
(b 100 nm), which lead to specific dose-dependent reduction in serum
cholesterol and LDL. Importantly, i.v. administered SNALP selectively
induced gene silencing only in the liver consistent with tissue BD pattern
[17]. Similarly, another study showed that SNALP, based on optimized
derivative of the ionizable cationic lipid DLinDMA, successfully
knocked-down Transthyretin in non-human primates with ED50 of
0.3 mg per kg body (mpk) in specific and durable manner [98].
SNALPs containing pooled siRNA against Zaire Ebola virus (ZEBOV)
RNA polymerase were reported to specifically inhibit ZEBOV replication
and increase survival in non-human primates [108], establishing a core
for RNAi-based bio-defensible tool against viral infection.
4.1.3. Lipidoids
Lipidoids are non-glycerol-based cationic lipid particles synthesized by conjugation of amines to acrylate or acrylamide [97].
A study exploiting accumulation of lipidoids in the MPS upon systemic administration was conducted in both non-human primates
and rodents and showed potent and specific dose-dependent gene
silencing of different genes in lipidoid-formulated siRNA system
[97]. In the first multitargeting study in vivo, a pool of five siRNA
sequences targeting hepatocytes were formulated with epoxidebased lipidoids (80 nm) and was delivered into mice. The study
showed effective simultaneous gene silencing of all the targeted
genes upon single injection. The same NP formulation was utilized
in the same study for potent silencing of transthyretin (TTR) in
non-human primates at 0.03mpk dose [109]. The advantage of this
approach is that it provides an opportunity for targeting multifactorial diseases by using a pool of several siRNA constructs.
4.1.4. Polyplexes
Polyplexes are polycation-based NPs (100–400 nm) formulated
siRNAs. The major advantage of using polycations is their structural
flexibility. Widely used polycations include biocompatible natural
(e.g. chitosan, Atelocollagen, cyclodextrin) as well as synthetic
(e.g. Polyethylenimin (PEI), poly (L-lysine) (PLL)) polymers
[110,111] (Fig. 1F, H).
Upon the use of polyplex-formulated LNA-siRNA Gao et al.
reported an extension in circulatory t1/2 and a reduction in renal
clearance compared to naked siRNA. Their work also demonstrated
the importance of selecting NP features for efficient targeted drug
delivery, as different polyplex formulations affected differently the
BD pattern as well as the PK characteristics of the delivered siRNA, a
significant aspect that could be utilized for targeted delivery. PEI is a
polycation used for RNAi delivery by self-assembly of the polymer
with siRNA molecules; however, PEI polyplexes bind nonspecifically
to cells and adsorb serum proteins, making their use for siRNA delivery inefficient, hence, conjugating PEG groups to PEI nanoplexes or
alternatively fine tuning the ratio of siRNA/PEI, as was well demonstrated in this study, can improve their delivery upon systemic
administration. The study conducted in healthy mice shows that i.v.
injection of JetPEI/LNA-siRNA polyplexes triggers siRNA accumulation
in the lung and to a smaller degree in the kidney and liver. The accumulation in the lungs could be explained by electrostatic interactions
between the not-fully neutralized polyplex and negatively serum
components, which lead to physical entrapment within the relatively
small pulmonary capillaries were the blood flow is slow [50]. Alternatively, this opsonization can trigger engulfment by immune cells in
the RES organs. Furthermore, electrostatic interactions and consequently adhesion could happen between the positively charged
polyplex and the negatively charged endothelium lining the lung,
kidney and liver (mucoadhesion). On the other hand, using chitosan
polyplexes for LNA-siRNA delivery in the same system results in substantial accumulation of chitosan polyplex in the kidney reasonably
due to mucoadhesion to the lining of the kidney [50], a known feature
characteristic with chitosan polymers [112].
Atelocollagen is a safe biomaterial that is widely used for in vivo
delivery of RNAi molecules. The polyplex size could be monitored
by altering the ratio of Atelocollagen to siRNA, for example a polyplex
with 100–300 nm diameter can be obtained by using low concentrations of Atelocollagen (0.05%) [92,94,111]. Such formulation was
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Fig. 1. Representative targeted RNAi delivery approaches for in vivo use. RNAi molecule could be complexed with mAb/mAb fragments (A) or other targeting peptides (C) via
protamine or 9R-nonamer. Also, it could be direct-conjugated to cholesterol (G) or other small molecules (H), Aptamer (D) and polymers (F). Encapsulation of the RNAi molecule
within polymeric NPs (E) or liposomes/immunoliposomes (B) could be an alternative approach. Figure adapted and revised with permission from Ref. [134].
utilized for siRNA delivery to the liver in a murine liver metastasis
model of lung cancer. The study showed a durable and dosedependent inhibition and apoptosis of tumor growth along with
withdrawal in liver metastasis [94]. Another study has reported specific dose-dependent gene silencing of MCP-1 and reduction in infiltration of immune cells to inflamed tissues, hence inflammatory
inhibition in murine ear model of contact hypersensitivity (allergic
dermatitis) [92]. Atelocollagen polyplexes were selectively delivered
to target sites and taken up by inflamed target cells, rather than
taken up by migrating inflammatory cells reasonably due to the EPR
effect in inflamed tissue.
The difference in BD and PK profiles of the various polyplexes
reflects the diversity in net charge, hydrodynamic diameter as well
as the shape of the NP, features that could be utilized for more efficient gene silencing.
4.1.5. Carbon-nanotubes (CNTS)
Carbon-nanotubes (CNTs) are nonspherical, fibrilous nano-cylinders
composed of single (1D single-walled CNTs, SWCNTs) or multiple
(multi-walled CNTs, MWCNTs) graphine layer(s) with length ranging
from 50 nm to 100 mm and diameter of 1–5 nm or 10–100 nm, respectively. Flexibility of CNT layers facilitates multi-valence binding to cells
and conjugating to targeting molecules [113,114]. Proper functionalizing
of CNTs by covalent or non-covalent strategies (such as coating with PEG
polymers or Tween-20) facilitates their solubility in aqueous solutions
and prevents non-specific interactions, thus minimizing toxicity
observed in the case of non-functionalized raw particles and increases
biocompatibility and circulating t1/2 [113,115].
The shape characteristics of the CNT could significantly affect its
BD. Whereas small spherical NPs and globular proteins with hydrodynamic diameter ≤ 6 nm are readily cleared by the kidney [46], length
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and shape of the NP should be taken into account when it comes to
the well-individualized cylindrical CNTs. For instance, functionalized
CNTs with diameter 1–30 nm and length of 200–2000 nm may exhibit rapid renal clearance and effective excretion from the body [115],
on the other hand, bundles-containing CNTs with same dimensions
that are not adequately individualized or undergo aggregation
in vivo cannot cross the glomerular filters of the kidney thus accumulate in the MPS organs or in the glomerular capillaries, respectively
[114,115].
Despite their advantages, the non-biodegradable characteristic of
CNTs makes their use for therapeutic purposes more challengeable and
requires further study. In a proof-of-concept study of SWNT-formulated
siRNA delivery to tumors in vivo, functionalized positively charged
SWCNTs (SWCNTs+) were conjugated to human telomerase reverse
transcriptase (hTERT) siRNA. The SWNT-formulated siRNA was injected
intratumorally and induced reduction in hTERT mRNA and protein levels
leading to inhibition in tumor cells growth in a xenograft mouse model
[40]. In a recent study by McCarroll et al. [116], the researchers have
developed SWNTs functionalized with lysine-based dendrimers covalently attached to lipid chains (Tol 7), while the positively-charged dendrimer
served for siRNA binding, the lipid moiety masks the hydrophilicity of
siRNA and facilitates cell binding. This system was utilized for systemic
delivery of anti-ApoB siRNA and showed effective reduction in ApoB
mRNA and protein levels, which led to reduction in serum cholesterol
with no evidence of toxicity or immunogenicity.
4.2. Active, target-specific RNAi delivery
Targeting molecules highly expressed on the cells' surface in the disease site of action is an attractive strategy for specific delivery while
improving PK and minimizing non-specific delivery to bystander
tissues, hence allowing the use of minimal therapeutics doses and
reducing off-target effects. Antibodies, natural ligands, ligand mimetics
and aptamers can be attached to or complexed with free or NPformulated siRNA constructs (Fig. 1) to enhance stability, induce cellular uptake as well as promote targeted delivery. It is important to note
that these targeting molecules must undergo internalization and specific endocytosis in order to obtain gene-silencing [86]. In the introduced
studies, the researchers validated targeting moieties' functionality
upon conjugating as well as specific binding by conducting in vitro
assays before systemic delivery.
4.2.1. Peptide-based targeting moieties
When conjugated directly to free siRNA molecules or to NPs,
monoclonal antibodies (mAbs) and other proteins composed of
targeting peptide and nucleic acid-binding domains could serve as
targeting moieties for systemic delivery of RNAi molecules. These
moieties are of crucial importance as they induce specific binding to
and receptor-induced internalization into the targeted cells. Ligands
overexpressed in disease-related tissues, such as integrins and other
extra-cellular matrix (ECM) receptors in cancer and inflammation as
well as growth factors receptors provide golden targets for those NPs.
Immuno-NP is an excellent strategy for specific targeted delivery.
However, conjugated mAB is likely to promote nonspecific interactions upon systemic administration (e.g. recognition by immune
cells via Fc receptors). Alternatively, engineered Ab fragments, such
as Fab and scFv, are safer and favorable in this case, albeit less stable
compared to the whole antibody, which lowers the NP t1/2 [86,120].
Zheng et al. succeeded in generating immunoliposomes that selectively target dendritic cells (DCs) [117]. The PEGylated CD40 siRNAcontaining immunoliposomes (siILs) are conjugated to NLDC-145, a
DC-specific mAB, and contain the cationic DDAB that facilitates siRNA
encapsulation with averaged hydrodynamic diameter of 86 nm. Upon
i.v. injection NLDC-145 siILs have shown to accumulate in spleen and
liver possibly due to specific targeting of DCs that are abundant in these
organs, whereas naked siRNA has accumulated mainly in kidney. When
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RNAi efficiency was examined, both naked and NLDC-145 siILencapsulated CD40 siRNAs induce gene silencing in splenic DCs 48 h
post injection (p.i.), however only siIL-encapsulated siRNA induced
prolonged specific silencing that lasted at least 12 days p.i. This observation can be explained by slow release of stable siRNA from siILs upon
spleen accumulation hence increasing its t1/2, underlying another advantageous feature of siRNA complexation over the degradable naked siRNA.
By utilizing integrins as excellent receptor targets for RNAi
delivery, Peer et al. have reported specific gene silencing of CyclinD1
(CyD1), a pivotal cell regulator, and inhibition of inflammation in an
experimental murine model of colitis by developing 100–200 nm
integrin–targeted and stabilized NP (I-tsNP) system encapsulating
siRNA-protamine complex (Fig. 1B). The positively charged small
nucleotide-binding protein protamine neutralizes and condenses
the negatively charged siRNA, protects it from degradation and aids
in its encapsulation within the NP [118]. Anti- β7 integrin was
attached to neutral lipid-based NPs coated with hyaluronan (HA), a
biocompatible and non-immunogenic glycosaminoglycan that
masks the NP from the immune system and increases its circulating
time in vivo [112]. Similarly, LFA-1 I-tsNPs were applied in suppression of HIV infection in a humanized mouse model using siRNA
against CCR5, a crucial regulator in early stages of HIV infection.
SiCCR5/LFA-1 I-tsNP promoted specific and durable (up to 10 days)
gene silencing in lymphocytes and macrophages without inducing
IFN response and immunogenicity [119]. Another advantage of
such immuno-NP systems is achieved by the synergistic effect
obtained upon blocking integrin-mediated leukocyte migration via
mAB-integrin interaction.
Via fusing Fab antibody fragment (F105) against HIV-1 envelope
(gp160) to protamine (F105-P), Song et al. have designed antibodyprotamine fusion protein (Fig. 1A) for systemically delivering a
mixture of anti-oncogenic siRNAs into mice grafted with gp160expressing B16 melanoma cells. The F105-P complexed siRNAs selectively targeted gp160-B16 cells and suppressed tumor growth [120].
Based on this strategy, Peer et al. have reported a selective targeting
for activated leukocytes using Integrin LFA-1 single chain Fv (scFv)protamine fusion strategy recognizing the conformationally distinct
activated form of LFA-1 [121], an observation that holds promise for
specific in vivo gene silencing while avoiding delivery to normal
bystander cells (mainly leukocytes in this specific case) and mitigating off-target effects in general and global immunosuppression, in
particular.
Although no toxicity was reported in those studies, the immunogenic response issue should be taken into consideration when using
AB for systemic delivery. Other peptide-based agents that have target
selectivity whilst exhibit less immunogenicity can be applied in RNAi
molecules targeted delivery.
Arg–Gly–Asp (RGD) peptides have been utilized for targeting αv
integrins expressed on many tumor vasculature. Comparable
sequences have been shown to recognize other isoforms of integrins
[131]. Attaching an RGD peptide to the PEGylated PEI (RPP) nanoplex
facilitated its delivery to tumor tissues. It has been reported that i.v.
delivered 70–100 nm RPP nanoplex self-assembled with VEGFR2siRNA selectively targeted tumor vasculature overexpressing integrins
and inhibited VEGFR2 expression leading to repression in tumor angiogenesis and decrease in growth rate in a murine tumor model [122].
Although CNT-based targeted delivery is still only in its infancy and
yet there is no published study regarding targeted systemic delivery
of CNT-formulated siRNA, Liu et al. utilized the stabilized, aqueous suspensions of SWCNTs coated with PEG chains adsorbed to phospholipids
(PL-PEG, diameter of 1–5 nm, length of 100–300 nm) that developed
by Kam and Dai [132] to conjugate RGD peptide (PL-PEG–RGD) and
study their BD by microPET scans 24 h post i.v. administration in a
xenograft murine model bearing tumor cells highly expressing αv
integrins [114]. SWNT conjugates exhibited slow excretion rate and
were mainly taken by the MPS (liver and spleen). The PEGylated
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SWNT conjugate with the longest PEG chain (PL-PEG5400) exhibited
lower uptake level by the liver consistent with their relatively higher
circulating t1/2 compared to PL-PEG2000, probably due to higher hydrophilicity and lower non-specific interactions with serum proteins leading to reduction in opsonization. However, when RGD was conjugated
to PL-PEG5400 a significant rapid uptake by tumor tissue was obtained
reasonably due to specific and targeted delivery of the PL-PEG–RGD
SWCNT conjugates and multivalency effect of binding multiple RGD
molecules onto the surface of SWCNT backed up with extending circulation time obtained by PEG-mediated MPS escaping. Within the period
of 24 h p.i. no toxicity or immune response were observed [114].
A peptide derived from rabies virus glycoprotein (RVG) was used
in transvascular siRNA delivery to the central nervous system
(Fig. 1C). RVG binds specifically to Acetylcholine receptor (AchR)
which is highly expressed by neuronal cells, and was stabilized by
attaching 9 residues of Arginine (9R nonamer) that also enables conjugation to and enhance cellular uptake of siRNA. When anti viral
siRNA (siFvE J) was complexed with RVG-9R peptide it has reported
to protect against fatal viral encephalitis in mice without triggering
immunogenicity [123].
4.2.2. Aptamer/siRNA chimeras
Aptamers are nucleic acid-based molecules that are used for diagnostic application as well as gene expression regulation and gene therapy. The RNA-aptamers/siRNA chimera is recognized and processed by
DICER generating 21–23 nt siRNA fragments in a miRNA-like manner,
this along with aptamer specific binding to target ligands, small size
which allow better tissue internalization and amenability for modifications, make it an appropriate strategy for induction of targeted RNAi
delivery in vivo. Additionally, this strategy is considered to be advantageous over protein-based targeted moieties in terms of cost, low immunogenicity and production complexity [31,124].
A10 aptamer binds specifically to PSMA (prostate-specific membrane antigen) on the surface of prostate cancer cells and tumor vascular endothelium, and has been shown to specifically inhibit tumor
growth when conjugated to siRNAs against anti-survival genes (Plk1
and Bcl2) overexpressed in most human tumors in a xenograft
model of prostate cancer. Interestingly, when A10-NP was examined
in another model where the tumors do not express PSMA, no tumor
regression was obtained demonstrating the targeting selectivity of
this system. This feature is worth note, particularly when triggering
apoptosis as in the current case, as it is of great importance not to target healthy cells [124]. In another study, a dual inhibitory function
anti-gp120 aptamer/siRNA chimera, in which both aptamer and
siRNA portions directed against HIV genes, was used to specifically
target cells expressing HIV receptor gp120 leading to repression of
HIV replication in tissue culture tests. An attractive approach for
RNAi induction that could be utilized for systemic delivery [125]
(Fig. 1D).
4.2.3. Other small molecules-siRNA NPs
An siRNA-delivery system (120–150 nm) that employs characteristics of both cationic LNPs and polyplexes was developed in the lab of
Huang [126]. siRNA constructs were mixed with calf thymus DNA
before complexing with protamine followed by coating with cationic liposomes (DOTAP and cholesterol), the liposomes-polycation-DNA
(LPD) was then mixed with a PEGylated ligand lipid (PEG–Anisamide–
DSPE). Anisamide ligand binds to sigma (σ) receptors highly expressed
by tumor cells. In one study conducted in a mouse xenograft tumor
model Anisamide-NPs systemically administrated were delivered mainly
to the tumor tissue and induced specific gene silencing of the EGFR in
addition to cell apoptosis and slowed down tumor progression.
Reduced liver uptake was also reported, probably due to relatively
reduced MPS delivery upon PEGylation. The calf thymus DNA serves
as a carrier that reduced particle size and increased delivery efficiency.
Immunostimulatory effects were observed in siRNA-NPs and thought
to be related to siRNA-immunostimulatory sequences, as empty NPs
(without siRNA) showed minimal immunogenic effects albeit these
DNA fragments contained immunogenic CpG motifs [126]. Similarly,
Lipotrust liposomes that consist of cationic lipid DC-6-14, cholesterol
and DOPE attached to vitamine A were used to encapsulate and deliver
anti-gp64 siRNA (VA-lip-siRNA gp46, ~150 nm) to hepatic stellate (HS)
cells, which overexpress retinol binding protein (RBP) that takes up
vitamine A from the circulation in progressive and lethal model of
liver fibrosis in rats. Upon i.v. administration of VA-lip, anti gp-46
siRNA led to collagen inhibition in HS cells and cell apoptosis mitigating
liver fibrosis and extending rat survival with no immnuostimulatory
effects [127].
Cyclodextrin polycation (CDP) self-assembles with and condenses
siRNA molecules to generate ~50–70 nm NPs stabilized by PEGylation.
Human transferring (Tf)-CDP-siRNA NPs were used for targeting metastatic cells overexpressing Tf-receptor in murine model of Ewing's family of tumors—EFT (mesenchymal malignancy in bone and soft tissues).
These conjugates have shown localization in and uptake into tumor
cells that led to transient reduction in tumor growth [128] (Fig. 1E).
Whereas Tf-CDP-NPs showed no significant immunostimulatory effects
upon i.v. administration into mice, the same system induced modest
pro-inflammatory cytokine and IFN productions as well as kidney and
liver toxicity in non-human primates. Those undesired side effects can
be overcome by injection of multiple lower i.v. doses [133].
siRNA dynamic polyconjugate (DPC) is a PEGylated nanosystem
composed of targeted-ligand agent linked by the reversible maleamate
linkage to the endosomolytic agent PBAVE (Poly butyl and amino vinyl
ethers) in which the siRNA is attached to PBAVE by reversible disulfide
bond (Fig. 1F). Attaching the ligand agent N-acetylgalactosamine (NAG)
has been reported to selectively target hepatocytes and not the
nonparenchymal cells (or the immune Kupffer cells) in the liver, whereas attaching mannose redirected the DPC to nonparenchymal cells away
from hepatocytes [129]. NAG DPCs (~10 nm) with anti-apoB or
anti-peroxisome proliferator-activated receptor alpha (ppara) siRNAs
induced specific and durable (t1/2 between 7 and 10 days) gene silencing selectively in the liver, which led to reduction in serum cholesterol
and ApoB levels and serum triglycerides, respectively. Moreover, the
NAG-mediated selective targeting to hepatocytes along with DPC's relative small size eliminates Kupffer cells-mediated toxicity in the liver
upon systemic administration of siRNA DPCs [129].
Generating cholesterol-siRNA (chol-siRNA) by conjugating cholesterol to 3′ end of the sense strand of siRNA constructs has been shown
to improve in vivo PK features (reducing circulation t1/2 and reducing
clearance from blood system) compared to unconjugated siRNAs
[130] (Fig. 1G). Utilizing this system with anti-apoB siRNA led to specific delivery to the liver and the jejunum and to a reduction in the
serum lipoprotein and the cholesterol levels in a transgenic mouse
model [130]. Similarly, cholesterol-conjugated antagomir122, against
miR-122, a liver-specific miRNA, led to efficient and durable (up to
23 days) silencing of this specific miRNA in mouse liver upon i.v.
administration [13]. Antagomir-122 was well-tolerated and regulated
the expression of genes involved in cholesterol biosynthesis and
reduced cholesterol levels in a specific manner.
5. Intracellular delivery of NP-associated RNAi molecules
A crucial requirement of efficient systemic siRNA delivery is the
ability to mask their net negative charge that consequently minimizes
aggregation and recognition by the immune system leading to an
increase in circulating t1/2 and most importantly facilitating binding
to target-cell membrane. Beyond selective delivery to the intended
cell type in the site of action, sufficient binding strength between
the NP and the cell surface along with efficient delivery to the appropriate sub-cellular compartment(s), are also required for targeted
gene silencing.
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5.1. Delivery to the cell membrane
Particle internalization into the cytoplasm upon cell-membrane
binding occurs by direct delivery through the cell membrane and
passive diffusion within the cytoplasm, or by energy-dependent endocytosis (Fig. 2). Because of the high protein concentration and viscosity within the cytoplasm, passive diffusion is slow and restricted to
NPs smaller than 30 nm [135]. Besides phagocytosis, that is restricted
to phagocytes, endocytosis in mammalian cells occurs through
diverse pinocytosis-based mechanisms (e.g. clathrin-dependent and
clathrin-independent caveole-mediated pathways). Phagocytosis
pathway involves endocytosis of particles larger than 1 μm [136]
these dimensions are beyond the scope of this review.
Targeted-NPs interact with the plasma membrane via specific cell
surface receptors/binding molecules, followed by engulfment in membrane invaginations forming the early endosome, which fuse to more
specialized vesicles (e.g. late endosomes, lysosomes, perinuclear vesicles). Finally, the cargo is delivered to distinct intracellular compartments or recycled to the ECM [137].
Clathrin-mediated endocytosis (CME) (Fig. 2.2) is a wellcharacterized internalization pathway in mammals by which the
engulfment of the cargo occurs through clathrin-coated pits formation
and follows the traditional abovementioned pathway of endocytosis.
NPs endocytosed via the CME experience a gradual drop in pH while
trafficked from early endosome (pH 5.9–6) to lysosome (pH 4.5–5.5)
through the late endosome (pH 5–6) as well as an exposure to nucleases that result in siRNA degradation. In the clathrin-independent
Caveolae-mediated endocytosis (Fig. 2.1) however, the engulfed NP is
trafficked through caveosome route, which increases its likeliness to
escape the drop in pH (intra-caveosome pH is neutral) and avoid nuclease degradation as lysosome trafficking does not occur in the caveolae
pathway [138,139].
Whether the NP internalization follows the CME or Caveolaemediated pathway is dependent on the NP physical features. Whereas
200–500 nm NPs are endocytosed mainly via caveolae-mediated
pathway, CME pathway is favored in the case of NPs ≤ 200 nm
[140]. NP charge and ligand moiety attached to it may also determine
the route of endocytosis [141]. Cationic NPs interact with the negatively charged cell membrane leading to internalization via fusion
route [142] and localization of the payload into the cell [143]. This
promising strategy for direct delivery to the cytoplasm does not override the potential for positive charge-mediated off-target and
undesirable effects. Therefore, targeted neutral NPs are more tolerable for systemic delivery, albeit endosomal entrapment must be
overcome.
5.2. Endosomal escape
In order to achieve efficient gene regulation, the NP must release
the siRNA molecules within the cell cytoplasm where they suppose
to enter the RISC and initiate gene silencing. Several strategies have
been developed for triggering endosomal escape of the cargo following CME before reaching late phases of endocytosis (i.e. trafficking to
late endosome and lysosome).
The bilayer structure of the biological membrane grants its stability and impermeability of large and hydrophilic molecules. The ability
of lipid-based system to promote inverted hexagonal (inverted
micelle-like) structure, instead of bilayer or lamellar phase is utilized
for destabilizing biological membrane and enhanced intracellular
delivery. Fusogenic lipids such as PEs as well as cationic lipids promote lamellar-to-inverted hexagonal transition which enhances
fusion with and interruption of the endosome membrane at acidic environments, hence are widely used for drug-delivery approaches
[104,137,144]. Considering the cationic lipids, the more the hydrophobic domain of the cationic lipid is unsaturated, the more fusogenic
the lipid is [100]. Cationic amino lipids-based NPs that are composed
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of ionizable amine headgroups with a pKa of 6–8 were studied for
drug and siRNA delivery [98,145]. At pH below their pKa (as in
endosomal compartments), the amine headgroups are fully protonated enhancing interaction with the endosomal/lysosomal membranes
and lead to membrane rupture and escaping to cytoplasm. On the
other hand, partial ionization of the amine headgroups at physiological pH allows for minimized interaction with serum proteins thus
reducing cytotoxicity. Ionizable amino lipids are composed of a
cholesteryl ether tail and an aliphatic tail. The use of longer aliphatic
tail in these lipids or increasing the amino lipid concentration results
in a decrease of lamellar to inverted-hexagonal transition temperature. It is worth noting that using amine headgroups with lower pKa
values enhances phase transition at higher pH values (i.e. in early
endosomes or even at cellular uptake phase) [145].
Endosomal swelling is a non-contact strategy for endosomal
escape. When delivered to endosomes, NPs with high buffering
capacity prevent acidification of the endosome (by absorbing protons) which lead to passive chloride ions (Cl -) influx into the endosome followed by osmotic swelling and eventually rupture of the
endosome membrane allowing cargo release to the cytoplasm. Cationic polymers, in particular tetratable polyamines, such as PEI,
which contains highly protonable amino groups have been reported
to act as “proton sponge” [146], and enhance nucleotide release to
cytoplasm [147]. “Proton sponge” mechanism occurs with other
polyplexes and cationic polymers, such as the amphiphilic NPs that
Wang et al. have designed. These ~ 130 nm NPs are composed of
polymerizable cysteine residues, “proton sponge” and hydrophobic
domains. The “proton sponge” domain triggers pH-sensitive membrane rupture in the endosome facilitating NP/siRNA escaping to the
cytoplasm. The cysteine residues and hydrophobic domains enhance
stable complexation of the NP with the siRNA as well as facilitate
siRNA dissociation from the NP via reduction of disulfide bonds by
and hydrophobic interaction with cytosol components [148].
Another non-contact strategy for endosomal escape is the use of
pH-sensitive lipoplexes/polyplexes. A hydrophilic pH-sensitive PEGylated
conjugate (POD) was attached to DOPE liposomes. While the POD
conjugate is stable at neutral pH, at acidic condition (below pH 5) the
POD is completely degraded. Equivalently, POD/DOPE liposomes have
been shown to remain stable at neutral pH for up to 12 h, however, at
pH 5.5 aggregation of the liposomes has been observed followed by
content release within 30 minutes [149]. In another study, PEGylated
PLL polyplex modified with the pH-sensitive endosomolytic Melittin
peptide (DMMAn-Mel) increases the efficiency of siRNA delivery [150].
Kam and Dai developed stabilized PL-PEG-coated SWCNTs with
PEG-amine groups (PL-PEG–NH2). The terminal NH2 group facilitated
the incorporation of siRNA molecules via disulfide bond, which in acid
conditions is cleaved by thiol-reducing enzymes and leads to payload
release [132].
6. NP-mediated RNAi: Clinical studies
Most of the RNAi-related advanced clinical studies are based on
localized delivery of naked RNAi molecules (Table 2), however,
systemically-applied RNAi research in non-human primate models
[17,97,98,108,133,151] hold promise for bringing it to a clinicallyrelevant stage.
Since the first RNAi-related clinical trials that introduced naked
siRNA intravitreal to patients with age-related macular degeneration
(AMD) and diabetic macular edema (DME) targeting vascular endothelial growth factor (VEGF) pathway, many approaches attempting
to deliver naked siRNAs in a local manner were conducted. Unfortunately, many of these approaches were terminated due to safety,
uncertain scientific achievements or companies' reorganization issues
[152,153]. Since then, more research was conducted in an effort to refine and improve this approach and to adapt it for systemic delivery
(Table 2).
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Fig. 2. Intracellular delivery pathways. NPs enter the cell membrane and trafficked through the cell cytoplasm via endocytosis pathways (1, 2) or passive delivery (3). Upon endocytosis NPs (1) are released from early endosomes and trafficked within the cytoplasm as in Caveolae-mediated pathway, or (2) trafficked via lysosomal pathway until escaping
from lysosome compartments near perinuclear region as in Clathrin-mediated endocytosis (CME) pathway. (3) Particularly small NPs exploit the passive diffusion within the cytoplasm. Figure adapted with permission from Ref. [135].
The first-in-human Phase I clinical trial involving systemic administration of targeted NPs for RNAi molecules delivery to solid tumor,
CALAA-01, was conducted by Davis et al. in 2008 [154,155]. CALAA-01
70 nm NP employs the Tf-CDP nanosystem previously described in
mice and non-human primates [128,133], with a number of modifications and refinements, in order to formulate the non-modified antiribonucleotide reductase subunit 2 (RRM2) siRNA [154]. Biopsies from
melanoma patients that undergo systemic administration of Tf-CDPNPs showed intracellular accumulation of the NPs selectively in tumor
tissues and not in adjacent epidermal tissues in a dose-dependent manner, in addition to reduction in both RRM2 mRNA and RRM2 protein
levels after several cycles of dosing [155].
Since then, seven candidates for systemic delivery were entered
the clinical Phase, six of them are SNALP-based systems. In 2008
SNALP-based tool was formulated with two siRNA constructs against
VEGF and kinesin spindle protein (KSP) and administered to patients
with liver cancer and transthyretin (TTR)-mediated amyloidosis
(ATTR). The ALN-VSP02 therapy was well-tolerated and induced
anti-VEGF and anti-tumor effects in most patients and continued to
second Phase I trial. In 2009 three candidates entered the clinical
Phase, two of which, ALN-TTR01 and TKM-ApoB are SNALP-based
therapies targeting the hepatocytes and formulated with antiTransthyretin (TTR) and anti-ApoB siRNAs in ATTR and Hypercholesterolemia patients, respectively. Post single dose, ALN-TTR01 led to
significant reduction in amyloid deposits in targeted tissues and in
serum TTR protein in a dose-dependent manner. Since patients who
received TKM-ApoB exhibited transient reduction in serum cholesterol levels, TKM-ApoB Phase I clinical trial was terminated. Moreover,
two of 17 patients exhibited immunostimulatory effects and elevated
cytokine production upon i.v. administration of the highest dosage of
the drug. In 2010, an improved related NP, TKM-PLK1, was clinically
tested in patients with advanced solid cancers and lymphoma that
are resistant to conventional therapeutics. This NP targets the pololike kinase 1 (PLK1) in an attempt to inhibit cell-cycle progression
and induce apoptosis in tumor tissue. Protein kinase N3 (PKN3),
which is an essential downstream component of the PI3K pathway
mediating metastasis of tumor cells was targeted in Phase I clinical
trial of Atu027 lipoplex in 2009 in patients with advanced colorectal
cancer, however, it is important to note that activation of the complement pathway was observed in Atu027 clinical trial, a finding that
necessitates further improvement of this delivery system. ALNPCS02 and TKM-EBOLA were recently entered Phase I clinical studies
for treatment of hypercholesterolemia and biodefense against Ebola
viral infection, respectively [153]. In 2011, two other SNALP-based
candidates have entered the clinical phase, TKM-EBOLA [108] and
ALN-PCS02 (Table 2).
The accumulated clinical data regarding ALN-VSP02 and Atu027
support for further development with the sponsors anticipating to
license the two compounds in 2012 [152] , this along with the
on-going and speeding up research concerning nanobiotechnology
is expected to establish for a revolutionizing and promising core for
new nano-based pharmaceutical market (Table 3).
7. Conclusions
The potential of RNAi molecules to silence genes in an allelespecific manner along with nanobiotechnology generate the basis
for a potent tool for validation and medical interventions in many
types of diseases such as cancer, inflammation, allergies and viral
infections. Per contra, our poor understanding of the biological
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Table 2
RNAi-based drug candidates in clinical studies (adapted and revised with permission from Ref. [152]).
Candidate
name
Disease
Target
Delivery system
Phase
Year
Bevasiranib
AMD
VEGF
Local-intravitreal needle injection.
2004
Cand5
ALN-RSV01
DGFi
TD101
AMD, DME
RSV infection
Acute kidney injury, delayed graft function
Pachyonychia congenita
Local-intravitreal needle injection.
Local-inhalation of unformulated sirRNAs
Systemic - naked sirRNA
Local-intradermal needle injection (skin)
QPI-1007
Chronic nerve atrophy, nonarteritic ischemic optic
neuropathy
Operable pancreatic ductal adenocarcinoma
VEGF
Viral RNA
p53
Mutant keratin
(K6a)
Caspase 2
III—was interrupted in
2009
II
II
II
I
Local-intravitreal needle injection
I
2009
Mutated KRAS
Local-drug elution
I
2010
Metastatic solid tumors.
Liver cancer, cancer with liver involvement
Advanced solid tumors
RRM2
VEGF, KSP
PKN3
I
I
I
2008
2008
2009
Hypercholesterolemia
ATTR
Solid cancers and lymphoma
Hypercholesterolemia
Ebola infection (biodefense)
ApoB
TTR
PLK1
PCSK9
Viral RNA
polymerase
Systemic-CDP NPs
Systemic-SNALP liposomes (hepatocytes)
Systemic-AtuPLEX lipoplex (vascular
endothelial cells)
Systemic-SNALP liposomes (hepatocytes)
Systemic-SNALP liposomes (hepatocytes)
Systemic-SNALP liposomes (solid tumors)
Systemic-SNALP liposomes (hepatocytes)
Systemic-SNALP liposomes (hepatocytes and
phagocytes)
I
I
I
I
I
2009
2009
2010
2011
2011
siG12D
LODER
CALAA-01
ALN-VSP02
Atu027
TKM-ApoB
ALN-TTR01
TKM-PLK1
ALN-PCS02
TKM-EBOLA
2004
2005
2007
2008
Abbreviations: ApoB: apolipoprotein B. AMD: age-related macular edema. ATTR: transthyretin (TTR)-mediated amyloidosis. CDP: cyclodextrin polycation. DME: diabetic macular
edema. KSP: kinesin spindle protein. NP: nanoparticle. PCSK9: proprotein convertase subtilisin/kexin type 9. PKN3: protein kinase N3. PLK1: polo-like kinase 1. RRM2: ribonucleotide reductase subunit 2. RSV: respiratory syncytial virus. siRNA: small interfering RNA. SNALP: stable nucleic acid lipid particles. VEGF: vascular endothelial growth factor.
pathways at the molecular and genomic levels as well as the toxicity
and tolerability-related issues concerning synthetic NPs may hamper
the clinical approval of highly specific, targeted and individualized
RNAi-based medicine. The emerging Omics area on the other hand,
provides more adequate and comprehensive understanding of the
specific mutant genes in diseases and the biological responses upon
NP-formulated RNAi delivery both in vitro and in vivo that would
aid in designing safer and more potent delivery strategies.
Beyond diagnostics, imaging methodologies and conventional
drug delivery systems, Nanobiotechnology field offers other exciting
tools for medical and therapeutic applications, such as the tissue
engineering discipline. This developing field intends to design
biocompatible scaffolds aiming to regenerate tissues as well as
resemble the ECM and provide a controlled release of the loaded
pharmaceutical agents upon the use of a wide range of biomaterials.
Recently, this strategy has been adapted for RNAi molecules delivery
mainly in in vitro and ex vivo studies [156]. A sustained and specific
RNAi molecules release is achieved upon incorporating NP-associated
RNAi molecules into scaffolds within the intended site of action,
allowing more efficient, specific and potent gene regulation as well
as mitigating several conventional delivery tools- related adverse
effects.
We believe that the combinatorial and inter-disciplinary approach
of RNAi, Nanobiotechnology and Omics era will pave the way for tolerable, targeted and highly effective personalized RNAi-based
nanomedicines, revolutionizing research, and turning RNAi to a new
therapeutic modality.
Table 3
Nano-based pharmaceuticals—expected global market size.
Nano-based pharmaceutical
2020 (US$ billions)
2025 (US$ billions)
Protein-based
Nucleic acid-based
Small molecules-based
14 ± 7
7±3
3±3
28 ± 14
14 ± 7
6±3
Based on pharmaceutical market mainly targeting cancer and inflammatory diseases
for therapeutic purposes (adapted with permission from Ref. [26]).
Acknowledgments
The authors wish to thank Ms. Varda Wexler for her help with the
graphics and illustrations.
This work was supported in part by grants from the Marie Curie
IRG-FP7 of the European Union, Lewis Family Trust, Israel Science
Foundation (award no. 181/10), the Kenneth Rainin Foundation, the
I-CORE Program of the Planning and Budgeting Committee and The
Israel Science Foundation (grant no. 41/11) and the FTA: Nanomedicines
for Personalized Theranostics of the Israeli National Nanotechnology
Initiative to D.P.
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