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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. Unauthenticated Download Date | 6/16/17 5:51 AM 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 Unauthenticated Download Date | 6/16/17 5:51 AM 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 Unauthenticated Download Date | 6/16/17 5:51 AM 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 Unauthenticated Download Date | 6/16/17 5:51 AM 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. Unauthenticated Download Date | 6/16/17 5:51 AM 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 Unauthenticated Download Date | 6/16/17 5:51 AM 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 References [1] Afonin KA, Bindewald E, Kireeva M, Shapiro BA. Computational and experimental studies of reassociating RNA/DNA hybrids containing split functionalities. Methods Enzymol. 2015;553:313-34. [2] Afonin KA, Bindewald E, Yaghoubian AJ, Voss N, Jacovetty E, Shapiro BA, et al. In vitro assembly of cubic RNA-based scaffolds designed in silico. Nat Nanotechnol. 2010 Sep;5(9):676-82. [3] Afonin KA, Cieply DJ, Leontis NB. Specific RNA self-assembly with minimal paranemic motifs. J Am Chem Soc. 2008 Jan 9;130(1):93-102. [4] Afonin KA, Desai R, Viard M, Kireeva ML, Bindewald E, Case CL, et al. 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