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DNA and RNA Nanotechnology 2016; 3: 1–10
Research Article
Open Access
Marina A. Dobrovolskaia*
Self-assembled DNA/RNA nanoparticles
as a new generation of therapeutic nucleic
acids: immunological compatibility and other
translational considerations
DOI 10.1515/rnan-2016-0001
Received February 8, 2016; accepted March 18, 2016
Abstract: Therapeutic nucleic acids (TNAs) are rapidly
being embraced as effective interventions in a variety of
genetic disorders, cancers, and viral/microbial infections,
as well as for use in improving vaccine efficacy. Many
traditional nucleotide-based formulations have been
approved for clinical use, while various macromolecular
nucleic acids are in different phases of preclinical and
clinical development. Various nanotechnology carriers,
including but not limited to liposomes, emulsions,
dendrimers, and polyplexes, are considered for their
improved delivery and reduced toxicity compared to
traditional TNAs. Moreover, a new generation of TNAs
has recently emerged and is represented by DNA/RNA
nanoparticles formed by the self-assembly of DNA, RNA,
or hybrid DNA-RNA oligonucleotides into 1D, 2D, and 3D
structures of different shapes. In this mini-review, I will
discuss immunocompatibility and other translational
aspects in the development of this new class of promising
nucleic acid therapeutics.
Keywords: nanoparticles, preclinical, immunotoxicity,
cytokines, anaphylaxis, complement, therapeutic nucleic
acids
*Corresponding author: Marina A. Dobrovolskaia, Nanotechnology
Characterization Laboratory, Cancer Research Technology Program,
Leidos Biomedical Research, Inc., Frederick National Laboratory
for Cancer Research, NCI at Frederick, Frederick, MD 21702, E-mail:
[email protected]
1 Introduction
Therapeutic nucleic acids (TNAs) provide exciting
opportunities for correcting disorders that would
otherwise be considered untreatable by conventional
small-molecule and protein therapeutics. Traditional
TNAs include small and macromolecular nucleic acids
that directly or indirectly interfere with DNA replication,
repair, and RNA synthesis; affect the stability of DNA or
RNA; perturb the flow of genetic information at the DNA,
mRNA, and protein levels; and stimulate the immune
system, causing either suppression of disease-specific
genes or stimulation of gene expression in response to an
antigen. Recently, a new generation of TNAs composed
of DNA, RNA, or hybrid DNA-RNA oligonucleotides
capable of self-assembling into nanosized structures has
emerged (1-12). For the purpose of this mini-review, I will
generally refer to this class of TNAs as self-assembled
DNA/RNA nanoparticles. When I discuss specific cases
reported for a single type of oligonucleotide or for hybrid
oligonucleotides, I will use more specific terminology
and call the cases RNA-, DNA-, or DNA-RNA hybrid
nanoparticles.
The self-assembled DNA/RNA nanoparticles vary
in shape. For example, the diversity of geometric
conformations, including squares, cubes, rings,
polyhedrons, jigsaw puzzles, branches, and filaments, have
been rationally designed and described by various groups
(2, 9, 10, 13-16). In addition, a new computational approach
developed by Shapiro et al. allowed for the controlled
design and assembly of DNA/RNA nanoparticles into 1D,
2D, and 3D structures (2, 17-20). The assembly of traditional
oligonucleotides into such nanostructures increases
their stability against thermal, radiation, chemical, and
enzymatic degradation without the need for additional
chemical modifications (20). For example, some of these
particles are resistant to degradation following high doses
© 2016 Marina A. Dobrovolskaia, published by De Gruyter Open.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.
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2 M.A. Dobrovolskaia
(up to 90 Gy) of 125I and 131Cs irradiation and to denaturation
in 8 M urea (12, 21). More importantly, oligonucleotides
composing self-assembled DNA/RNA nanoparticles can be
made interchangeable and swapped in and out of a particular
context without disturbing the overall nanoparticle
structure (22). This property allows using oligonucleotides
(DNA, RNA, or a hybrid DNA-RNA) not specific to the target
gene as a scaffold for delivery and controlled release of
gene-specific therapeutic oligonucleotides (e.g. siRNA,
antisense DNA oligonucleotide, ribozyme, aptamer, or
antisense RNA) (8, 22-26). Several strategies for forming
such complex nanoparticles have been described, and
they include single-strand RNA assembly, RNA-DNA hybrid
self-assembly, RNA architectonics, and co-transcriptional
assembly (22). The therapeutic efficacy of some of these
nanostructures has been described in several in vitro and
in vivo models of cancer and AIDS (7, 8, 20, 25-27). The
DNA /RNA nanotechnology arena is developing rapidly.
Understanding the critical path for translation of this
category of TNAs toward clinical applications is important
for implementing the benefits of this technology in patient
care.
Although requirements for TNA drug candidates
are the same as those for other drugs (they must be
stable in a biological matrix, potent, feature favorable
pharmacokinetics (PK) and pharmacodynamics (PD),
target specificity, and have strong safety profiles, while
also displaying efficacy against selected indication[s]),
and some of their general properties are similar to those of
small molecules and biologics, TNAs have several unique
aspects that place them into a separate category of drugs
(28). These unique features are related to their molecular
weight, structure, PK and absorption, distribution,
metabolism, and excretion (ADME), immunogenicity, and
off-target toxicity (28). Since the DNA/RNA nanoparticle
technology is still in early preclinical development, both
their properties that are shared with traditional TNAs
as well as their unique attributes are not completely
understood. Intuitively, due to the differences in geometric
shape, molecular weight, and secondary structure, selfassembled DNA/RNA nanoparticles may be assigned
to a separate category of drugs. In Table 1, I compare the
general properties of traditional TNAs, small-molecule
drugs, and biologics established by the Drug Information
Association Oligonucleotide Safety Working Group with
corresponding parameters of self-assembled DNA/RNA
nanoparticles, and introduce additional properties, such as
particle hydrodynamic size, which, while not being unique
to self-assembled DNA/RNA nanoparticles, is an important
determinant of their PK, ADME, and safety profiles.
The primary challenges for selecting an indication for
traditional TNAs stem from the limited success of smallmolecule or protein therapeutics and the administration
to tissues and cell types with favorable PK/PD responses
to nucleic acid–based agents. As such, many traditional
TNAs are directed against targets in the liver, kidneys,
eyes, and skin. While considering target selection for selfassembled DNA/RNA nanoparticles is largely similar to
that of traditional TNAs, the main indication extensively
explored so far is cancer, closely matching the indication
profiles of other nanotechnology-based formulations
(29). Similar to traditional TNAs, productive uptake
into the appropriate tissue, cell type, and intracellular
compartment is vital for the therapeutic success of selfassembled DNA/RNA nanoparticles.
Challenges related to preclinical and clinical
translation of traditional TNAs have been described before
(30-32). Herein, I will summarize the immunological
and hematological toxicities hampering translation of
traditional TNAs, and focus on translational challenges
for self-assembled DNA/RNA nanoparticles, with an
emphasis on their immunocompatibility.
2 Traditional Therapeutic Nucleic
Acids
Small-molecule TNAs include nucleotides and
nucleosides. Many of these products are used in the
clinic to treat viral infections and cancer (33, 34).
Macromolecular TNAs are represented by structurally
and chemically diverse products, including three
generations of antisense oligonucleotides (ASOs), triplexforming oligodeoxyribonucleotides, immunostimulatory
oligonucleotides, catalytic oligonucleotides, inhibitory
DNA, inhibitory RNA, plasmid DNA, aptamers, and
mRNA (34). First-generation ASOs include products
based on both natural (phosphodiester) and chemically
modified (phosphorothioate) backbones. The secondgeneration phosphorothioate ASOs are characterized
by chemical modifications of the 2′ site of the sugar
moiety to augment stability in vivo, whereas the thirdgeneration phosphorothioate ASOs contain much more
diverse modifications, including chemical alterations of
the native backbone, incorporation of internal bridges
within each backbone sugar (locked nucleic acids),
and even complete disruption of the sugar backbone
(peptide nucleic acids). These TNAs are considered for
the treatment of a broad spectrum of disorders resulting
from abnormal gene function, which include, among
others, genetic diseases, autoimmune diseases, and
cancer. Various concerns related to TNA stability in
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Translational considerations for DNA/RNA nanoparticles 3
Table 1. Comparison of the general properties of traditional low–molecular weight drugs, nucleic acids, and biologics with those of
self-assembled DNA/RNA nanoparticles. SAR – structure–activity relationship; PK – pharmacokinetics; ADME – absorption, distribution,
metabolism, and excretion; CMC – chemistry and manufacturing control; NA – not applicable; NID – not investigated in sufficient detail;
* – certain attributes summarized for these drugs are based on the Drug Information Association Oligonucleotide Safety Working Group
and reproduced from reference (28); ** – the average size of therapeutic proteins is >30 kD; the size range of therapeutic peptides is much
lower; a range is given to reflect the heterogeneity of this class of therapeutics; *** - these data is based on reference (78).
Attribute
Small Molecule*
Nucleic Acid*
Self-assembled DNA/RNA Biologics*
Nanoparticles
Molecular weight
Hydrodynamic size
Manufacture
<1kD
NA
Chemical synthesis
~6-7 kD
<1 nm
Chemical synthesis
~80-200 kD
5-30 nm
Biochemical synthesis
Structure
Single entity, high purity
Immunogenicity
Single entity with some
product-related impurities
Intra- and extracellular
Intra- and extracellular
Database may be available Chemistry-specific class
effect
Species-specific metabo- Catabolized to nucleotilites; distribution to many des and other chemical
tissues
modification– dependent
metabolites; selected
tissue distribution
Never
Rare
Species specificity
Sometimes
Sometimes
Off-target toxicity
Often
Sometimes
Target distribution
SAR
PK/ADME
biological matrices, pharmacokinetics, and toxicity have
been described (31, 32, 35-40). Below, I will highlight
the immunotoxicity of traditional TNAs and common
strategies for overcoming this toxicity, including a
delivery option using nanotechnology carriers and the
immunological challenges associated with it.
2.1 Immunotoxicity
The undesirable toxicity of small-molecule TNAs affecting
normal functioning of the immune system include
both immunosuppressive and immunostimulatory
reactions. For example, myelosuppression, neutropenia,
thrombocytopenia, lymphadenopathy, suppression of
the immune response to common antigens, anaphylaxis,
delayed-type hypersensitivity reactions, autoimmune
hemolytic anemia, cytokine release syndrome,
systemic inflammatory response syndrome, anemia,
and thrombosis have been reported as toxicities for
drugs in this TNA category. The common spectrum
of immunotoxicities limiting preclinical and clinical
translation of macromolecular TNAs also includes the
induction of cytokines, chemokines, and type I and type
Complex, heterogeneous
~1-150 kD**
1.6-14 *** nm
Biological synthesis or
biologically derived
Complex, heterogeneous
Intra- and extracellular
Largely extracellular
Chemistry-, size-, and
No database
shape-specific class effects
NID
Catabolized to amino acids;
distribution is limited
NID, but may be greater
than that of nucleic acid
drugs due to larger size
NID, but expected to be
similar to nucleic acid
category
NID
Often
Generally species specific
Rare
II interferons; the stimulation of fever; the activation of
monocytes, T cells, B cells, natural killer (NK) cells, and
dendritic (DC) cells; the activation of the complement
system; prolongation of plasma coagulation time; as well
as variety of hematological changes (leukopenia, anemia,
thrombocytopenia, and lymphopenia). A more granular
description of these toxicities and their relation to certain
types of TNAs are available in earlier reviews (30, 32).
2.2 Strategies for reducing undesirable
immunostimulation
Strategies for reducing unwanted immunostimulation
from oligonucleotide-based drugs include patients’
premedication with immunosuppressive cocktails
containing dexamethasone (an immunosuppressive
agent), acetaminophen (an antipyretic agent),
diphenhydramine or certidine (histamine H1 receptor
blockers), and ranitidine (a histamine H2 receptor
blocker) (41). Additionally, slow infusion rates to avoid
high plasma levels of nucleic acid and administering the
drugs through non-systemic routes, minimizing contact
between the drug and immune cells in the blood, could be
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4 M.A. Dobrovolskaia
attempted (32). Various alternative approaches, including
chemical modification of nucleotides and backbones, and
delivery options have been investigated for improving the
toxicity of macromolecular TNAs (42-44).
2.3 Immunotoxicity challenges associated
with delivery methods
Delivery routes commonly used for administering
traditional TNAs include subcutaneous (s.c.) injections,
as well as intravenous (i.v.) and s.c. infusions. Rapid
distribution from the injection site to the plasma has
been extensively studied and described for two types
of traditional TNAs: phosphorothioate antisense DNA
oligonucleotides and siRNA (45-48). While unformulated
antisense DNA oligonucleotides rapidly distribute to
a variety of organs, siRNA is degraded within the first
minutes of distribution to the plasma (45-48). To protect
siRNA and other unstable TNAs from nucleases, and
improve their delivery to target organs, tissues, and cells,
various nanotechnology carriers have been considered,
including, among others, polymer-based carriers, lipid
vesicles and lipid-like structures, bolaamphiphiles,
polysaccharides, dendrimers, colloidal gold, and silicon
nanoparticles(49-56).
Formulating
oligonucleotides
with nanocarriers is typically based on electrostatic
interactions between an anionic oligonucleotide and a
cationic carrier. Noncovalent binding of an oligonucleotide
to a carrier is needed to provide drug release and efficacy.
Despite some benefits, noncovalent binding is often
associated with instability of the carrier–oligonucleotide
complex in the plasma, which may lead to premature
drug release and/or toxicity of the cationic carrier to
circulating immune cells and plasma proteins. Cationic
particles are known to be cytotoxic to a variety of cells,
including the cells of the immune system (57). Cationic
polymers and nanoparticles with cationic surfaces have
been shown to induce cytokine secretion, exaggerate
endotoxin-mediated toxicities, activate the complement
system, trigger leukocyte procoagulant activity, and bind
certain plasma proteins, causing subsequent changes
in the conformation and/or function of these proteins
(58-60). Several studies suggested that the failure of
the oligonucleotide base modification to eliminate
immunotoxicity of nanotechnology-formulated drugs was
due to the significant immunostimulatory contribution
from the nanocarrier itself (61). For example, cationic lipids
and components used to prepare liposomes, emulsions,
or micellar nanocarriers (2-(4-[(3β)-cholest-5-en-3-yloxy]
butoxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien1-yloxy]propan-1-amine [CLinDMA] with or without
cholesterol,
polyethyleneglycol-dimyristoylglycerol,
trimethylammonium propane-cholesterol, 1,3-dioleoyl-3trimethylammonium propane [DOTAP], and protamine)
have been reported to contribute to siRNA immunotoxicity
(61-64). An analysis of the literature reporting on cytokine
induction by adenoviral vectors versus lipid-based
nanocarriers suggested that liposomes and lipoplexes
exhibit greater immunostimulatory potential than viral
vectors (65). Despite the success of using nanocarriers
to overcome some challenges related to the PK and
general toxicity of traditional TNAs, addressing their
immunotoxicity still requires additional efforts, and some
strategies have been proposed before (42).
3 Self-assembled DNA and RNA
nanoparticles
3.1 Current delivery methods and routes of
administration
Self-assembled DNA/RNA nanoparticles are usually
administered by injection directly into the target tissue
(e.g. intratumoral and subconjunctival) or systemically
into circulation via i.v. injection (23, 25, 26, 53, 66, 67). In
addition to optimizing DNA/RNA nanoparticle structural
complexity, introducing chemical modifications to the
backbone and individual nucleotides are commonly used
to improve the stability of DNA and RNA oligonucleotides
in biological matrices and to increase their circulation
time following systemic administration (33). Despite
the increased oligonucleotide stability and prolonged
circulation time, self-assembled DNA/RNA nanoparticle
delivery to target tissues and cells requires additional
approaches. These approaches are similar to those used
for traditional TNAs. Cationic lipids and bolaamphiphiles
have been reported in the current literature as the most
common ways of formulating self-assembled DNA/
RNA nanoparticles for in vivo applications (68, 69).
Lipofectamine, a cationic liposome-based transfection
reagent, is commonly used for experiments that require
self-assembled DNA/RNA nanoparticle delivery into target
cells in vitro (1, 4, 7, 23).
3.2 Plasma kinetics and distribution to
organs following systemic administration
To date, detailed pharmacokinetic studies of selfassembled DNA/RNA nanoparticles with various shapes
and compositions are limited. Available pharmacokinetic
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studies are usually conducted using fluorescent probes
(“beacons”), which have recognized limitations. The
mechanisms for chemical conjugation of such probes
to the individual components of self-assembled DNA/
RNA nanoparticles and their instability complicate
the interpretation of these studies. A comparison
of biodistribution profiles and clearance rates of
self-assembled DNA/RNA nanoparticles to those of
traditional TNAs using of the most common approaches
in the pharmaceutical industry (radioactive labeling
and class-specific dual hybridization ligand–binding
assays) is required. Moreover, studying nanotechnology
formulations requires monitoring both the carrier and
the active pharmaceutical ingredient (API). Stern’s group
has reported the importance of using dual labeling
to accurately monitor the distribution of both the
nanotechnology carrier and the API cargo (70). Complex
self-assembled DNA/RNA nanoparticles containing
an oligonucleotide-based scaffold as a carrier and a
therapeutic oligonucleotide (API) have so far not been
investigated using this approach. The examples described
below rely on studies utilizing fluorescent probes and,
as such, should be considered conclusive only for the
oligonucleotide moiety covalently linked to a fluorescent
dye. Following i.v. administration, three-way junction
(3WJ) pRNA nanoparticles were shown to distribute to
target tissues without significant distribution to other
organs, such as the liver, spleen, kidneys, or lungs, where
other types of nanoparticles typically accumulate (20, 21,
25, 27, 71). The distribution half-life of these self-assembled
RNA nanoparticles was estimated to be approximately
1 hour, and the terminal half-life was estimated to be
between 5 and 10 hours (71). Metabolites and excretion
routes were not studied, albeit based on the small particle
size (<11 nm), renal excretion route was hypothesized
(71). Another study demonstrated that tetrahedronforming oligonucleotide particles carrying siRNA had
a plasma half-life of 25 minutes (72). Twelve hours after
systemic administration, these particles were detected
in only the tumor and kidney, supporting a hypothesis
that renal excretion is the primary route of clearance
for these particles (72). The half-life and excretion route
of self-assembled RNA nanoparticles reported in these
studies are similar to those described in clinical trials
employing i.v. infusion of traditional macromolecular
TNAs (73). Afonin et al. reported the accumulation of
self-assembled DNA-RNA hybrid nanoparticles in tumors
within three hours of systemic i.v. injection (23). Besides
tumors, self-assembled DNA-RNA hybrid particles were
also detected in other tissues, such as liver, spleen,
Translational considerations for DNA/RNA nanoparticles 5
heart, lungs, brain, kidneys, and bladder (23). The
accumulation in the kidneys and bladder also suggests
a renal excretion of these particles; however, similar to
other studies, urine specimens from treated animals and
corresponding metabolite profiles were not investigated.
Increased stability, low-protein binding, and prolonged
circulation time were suggested to benefit the delivery
of self-assembled RNA nanoparticles to tumors via the
enhanced permeability and retention effect (25). To my
knowledge, no published study explores a relationship
between the sizes and shapes of self-assembled DNA/
RNA nanoparticles, as well as their distribution to tumors
with various degree of vascular leakage, in order to fully
understand the relevance of the enhanced permeability
and retention effect to this new category of TNAs. An
interesting finding was reported by Guo et al., who
compared the distribution of 3WJ RNA nanoparticles with
that of a parent RNA oligonucleotide and self-assembled
RNA nanoparticles forming a four-way junction (pRNA-X)
to the sclera and retina upon topical and subconjunctival
injection (66). Following topical administration, none
of the studied particles and controls distributed to the
eyes; whereas subconjunctival injection resulted in the
distribution of 3WJ pRNA and pRNA-X nanoparticles to
the conjunctiva, cornea, and sclera, while only pRNA-X
distributed to the retinal cells (66). The lymphatic system
was suggested to provide an excretion route for these
particles after subconjunctival injection (66). 3.3 Distribution into and within cells
Targeted delivery to the cell of interest is achieved by
attaching aptamers or small molecules to self-assembled
DNA/RNA nanoparticles. For example, folate-targeted
3WJ pRNA nanoparticles were shown to preferentially
accumulate in cells overexpressing the folic acid receptor
(20). Similarly, pRNA nanoparticles targeted with an HIV
gp120 protein–specific aptamer accumulated in cells
overexpressing this protein (74). After uptake by cells, selfassembled hybrid DNA-RNA nanoparticles were reported
to accumulate in the endosomal compartment (23). In
contrast to traditional TNAs, which were shown to be
mainly internalized via pinocytosis and podocytosis (32),
the mechanisms of self-assembled DNA/RNA nanoparticle
uptake by target cells have not been investigated in detail.
The role of protein binding and the mechanisms of selfassembled DNA/RNA nanoparticle uptake by the cells of
the mononuclear phagocytic system (MPS) also remain
largely unknown.
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6 M.A. Dobrovolskaia
3.4 General toxicity
While distribution of self-assembled DNA/RNA
nanoparticles to off-target tissues (in the liver, spleen,
kidneys, lungs, and/or brain) (23) has been documented,
the toxicity of particles in these organs is not yet
understood. Most papers describing self-assembled
DNA/RNA nanoparticles report that these constructs
are generally well tolerated in rodents (21, 26, 71).
Nevertheless, dose range finding toxicity studies designed
to evaluate maximum tolerated dose and target organ
toxicity using clinically relevant dosing regimen and route
of administration, in rodents and non-rodents are needed
to understand the toxicity of self-assembled DNA/RNA
nanoparticles.
3.5 Immunotoxicity
Similar to the general toxicity profile, the immunological
compatibility of self-assembled DNA/RNA nanoparticles
is not yet fully understood (5, 23, 75). Although 3WJ pRNA
nanoparticles did not induce pro-inflammatory cytokine
TNFα and interferon-dependent gene expression in KB
cancer cell line and murine RAW264.7 macrophage cell
line, and did not activate the expression of TLR3, TLR7,
and TLR9 in human peripheral blood monocytes, these
data are insufficient for making conclusions on the
immunostimulatory potential of the particles. Conclusions
cannot be made because culture supernatant analysis for
the presence of type I and type II interferons, and for a broad
spectrum of other pro-inflammatory cytokines in human
whole-blood and isolated peripheral blood mononuclear
cells (PBMC) was not performed, and because longer time
points (over 3 hours) were not analyzed (71). Intriguingly,
the same group reported that RNA nanoparticles with
different shapes induced pro-inflammatory cytokines
in vitro and in vivo in a shape-dependent manner (76).
Triangular-shaped self-assembled RNA nanoparticles
appeared to be more potent than the equivalent square or
pentagonal structures, while square self-assembled RNA
nanoparticles were more potent than their pentagonal
counterparts in inducing TNFα and IL-6 in mouse
macrophage RAW 264.7 models (76). When these particles
were used to deliver CpG DNA oligonucleotides, reverse
shape-effect relationships were observed: pentagonal
self-assembled RNA nanoparticles carrying several CpG
DNA oligonucleotides displayed the highest potency by
inducing higher levels of TNFα and IL-6 vs. their triangle
and square counterparts carrying CpG oligonucleotide
payloads (76). The biochemical foundation of these
differential shape-dependent cytokine induction effects is
yet to be elucidated and warrants additional mechanistic
studies. In another series of experiments, RNA nanocubes
were reported to be more immunostimulatory in human
PBMC and whole-blood cultures capable of inducing
higher levels of type I interferon and pro-inflammatory
cytokines than their parent RNA oligonucleotides
and DNA nanocubes of similar sizes (7). Afonin et
al. argued that the RNA nanocubes could benefit the
delivery of immunomodulatory oligonucleotides via
alternative routes (such as s.c., intradermally (i.d.), or
intramuscularly (i.m)) and could be used for indications
in which immune response stimulation is desirable,
while less immunostimulatory DNA nanocubes could
be used as drug delivery vehicles. The authors further
hypothesized that the higher pro-inflammatory potential
of RNA nanocubes is due to the activation of intracellular
receptors sensing nucleic acids (RIG1 and/or MDA5)
because type I interferon stimulation by RNA nanocubes,
but not by DNA nanocubes, required the expression of
the MAVS protein, an essential adaptor in the RIG/MDA
pathways (7).
When self-assembled DNA/RNA nanostructures with
pro-inflammatory properties are used for systemic delivery
of specific gene-targeting siRNA or DNA oligonucleotides,
additional approaches similar to those described above for
traditional TNAs may also be explored to avert detrimental
immunostimulation side effects.
3.6 Translational considerations
The flexibility of design strategies and overall versatility
of self-assembled DNA/RNA nanoparticles engineered
for oligonucleotide delivery with therapeutic modalities
have been studied extensively (14, 16, 18, 20-22, 25-27,
72). Translating these promising concepts into the
clinic requires additional investigations. Current gaps
in understanding the pharmacokinetics and toxicology
of self-assembled DNA/RNA nanoparticles include the
following research topics: local concentration of selfassembled DNA/RNA nanoparticles at the site of injection
and potential for systemic exposure to self-assembled
RNA/DNA nanoparticles when non-systemic routes of
administration are used; clearance rate, clearance routes,
plasma kinetics, distribution to organs after systemic (i.v.)
and alternative administration routes (s.c. i.d., i.t., etc.),
and dependence of these parameters on particle dose and
dosing regimen; the role of physicochemical parameters
(size, charge, secondary structure, conformation of surfaceexposed oligonucleotides, chemical modifications, and
the primary sequence of oligonucleotides composing
the core and therapeutic component of self-assembled
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DNA/RNA nanoparticles) in distribution to organs and
cell types; the relationship between tissue distribution
and plasma kinetics; protein binding and distribution
to cells and organs of the MPS; the mechanism of
metabolism and metabolic profiles; sequence- and shapedependent and -independent toxicity (especially the
effects on the kidneys and liver because these organs
were reported as primary target organs for traditional
TNAs); immunological and hematological compatibility
(especially the effects on the complement system, the
blood coagulation system, and the induction of cytokines,
chemokines, and interferons because these toxicities
were reported to be common dose-limiting factors for
traditional TNAs); identifying any class-related toxicities;
and understanding genotoxicity and reproductive effects.
If self-assembled DNA/RNA nanoparticles are formulated
using lipids, polymers, or other carriers, understanding
the role of these carriers on toxicity and the PK profile of
self-assembled DNA/RNA nanoparticles is also needed,
as many such carriers are not inert and may alter
biodistribution and contribute to toxicity (30, 42). One of
the common pitfalls in the contemporary nanomedicine
field is nanoparticle contamination with bacterial LPS,
with the subsequent ability of contaminated nanoparticles
to exaggerate endotoxin-mediated inflammation (77).
The translational challenges associated with endotoxin
contamination detection, quantification, and removal
from therapeutic formulations are fully pertinent to DNA/
RNA nanoparticles. It is unlikely that self-assembled
DNA/RNA nanoparticles will per se exaggerate endotoxinmediated inflammation, because this property was
attributed to the cationic particle charge, whereas selfassembled DNA/RNA nanoparticles are predominantly
anionic. However, whenever a cationic carrier is used for
self-assembled DNA/RNA nanoparticle delivery, this issue
will become a relevant risk factor to such formulations.
Endotoxins are also undesirable in self-assembled
DNA/RNA nanoparticles because they upregulate the
expression of proteins responsible for recognizing nucleic
acids, thus increasing the sensitivity of the immune cells in
recognizing nucleic acid–based particles. Finally, synergy
in inflammatory responses between self-assembled DNA/
RNA nanoparticles and endotoxins may also contribute
to the greater toxicity of endotoxin-contaminated selfassembled DNA/RNA nanoparticles. Potential sources of
endotoxin contamination and a strategy for minimizing it
during self-assembled DNA/RNA nanoparticle synthesis
have been recently described (5).
Translational considerations for DNA/RNA nanoparticles 7
4 Conclusion
DNA/RNA nanotechnology has unlocked tremendous
opportunities for both fundamental and applied
areas of chemistry and biology, and paves the way for
deploying innovative clinical strategies. Translating
novel self-assembled DNA/RNA nanoparticles into the
clinic will mandate additional investigations of their
pharmacokinetic and toxicology profiles. The previously
established and currently available knowledge of
traditional therapeutic nucleic acids thus offers an
essential stepping stone for detailed translational research
focused on self-assembled DNA/RNA nanoparticles.
ACKNOWLEDGMENTS: This study was supported by
federal funds from the National Cancer Institute, National
Institutes of Health, under contract HHSN261200800001E.
The content of this publication does not necessarily
reflect the views or policies of the Department of Health
and Human Services, nor does mention of trade names,
commercial products, or organizations imply endorsement
by the U.S. Government. I am grateful to Drs. Serguei
Kozlov and Stephan Stern for helpful discussion.
Conflict of interest: Authors state no conflict of interest
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