Break to Build: Isothermal Assembly of Nucleic Acid Nanoparticles (NANPs) via Enzymatic Degradation.

The intrinsic properties of RNA and DNA biopolymers emphasized by engineered nucleic acid nanoparticles (NANPs) offer accelerated development of next-generation therapies. The rational design of NANPs facilitates programmable architectures intended for regulated molecular and cellular interactions. The conventional bottom-up assembly of NANPs relies on the thermal annealing of individual strands. Here, we introduce a concept of nuclease-driven production of NANPs where selective digestion of functionally inert structures leads to isothermal self-assembly of liberated constituents. The working principles, morphological changes, assembly kinetics, and the retention of structural integrity for system components subjected to anhydrous processing and storage are assessed. We show that the assembly of precursors into a single structure improves stoichiometry and enhances the functionality of nuclease-driven products. Furthermore, the experiments with immune reporting cell lines show that the developed protocols retain the immunostimulatory functionality of tested NANPs. The presented approach enables exploitation of the advantages of conditionally produced NANPs and demonstrates that NANPs' stability, immunorecognition, and assembly can be regulated to allow for a more robust functional system.

RNA-DNA fibers and polygons with controlled immunorecognition activate RNAi, FRET and transcriptional regulation of NF-κB in human cells.

Nucleic acid-based assemblies that interact with each other and further communicate with the cellular machinery in a controlled manner represent a new class of reconfigurable materials that can overcome limitations of traditional biochemical approaches and improve the potential therapeutic utility of nucleic acids. This notion enables the development of novel biocompatible 'smart' devices and biosensors with precisely controlled physicochemical and biological properties. We extend this novel concept by designing RNA-DNA fibers and polygons that are able to cooperate in different human cell lines and that have defined immunostimulatory properties confirmed by ex vivo experiments. The mutual intracellular interaction of constructs results in the release of a large number of different siRNAs while giving a fluorescent response and activating NF-κB decoy DNA oligonucleotides. This work expands the possibilities of nucleic acid technologies by (i) introducing very simple design principles and assembly protocols; (ii) potentially allowing for a simultaneous release of various siRNAs together with functional DNA sequences and (iii) providing controlled rates of reassociation, stabilities in human blood serum, and immunorecognition.

Tagging and Capturing of Lentiviral Vectors Using Short RNAs.

Lentiviral (LV) vectors have emerged as powerful tools for transgene delivery ex vivo but in vivo gene therapy applications involving LV vectors have faced a number of challenges, including the low efficiency of transgene delivery, a lack of tissue specificity, immunogenicity to both the product encoded by the transgene and the vector, and the inactivation of the vector by the human complement cascade. To mitigate these issues, several engineering approaches, involving the covalent modification of vector particles or the incorporation of specific protein domains into the vector's envelope, have been tested. Short synthetic oligonucleotides, including aptamers bound to the surface of LV vectors, may provide a novel means with which to retarget LV vectors to specific cells and to shield these vectors from neutralization by sera. The purpose of this study was to develop strategies to tether nucleic acid sequences, including short RNA sequences, to LV vector particles in a specific and tight fashion. To bind short RNA sequences to LV vector particles, a bacteriophage lambda N protein-derived RNA binding domain (λN), fused to the measles virus hemagglutinin protein, was used. The λN protein bound RNA sequences bearing a boxB RNA hairpin. To test this approach, we used an RNA aptamer specific to the human epidermal growth factor receptor (EGFR), which was bound to LV vector particles via an RNA scaffold containing a boxB RNA motif. The results obtained confirmed that the EGFR-specific RNA aptamer bound to cells expressing EGFR and that the boxB containing the RNA scaffold was bound specifically to the λN RNA binding domain attached to the vector. These results show that LV vectors can be equipped with nucleic acid sequences to develop improved LV vectors for in vivo applications.

The Recognition of and Reactions to Nucleic Acid Nanoparticles by Human Immune Cells.

The relatively straightforward methods of designing and assembling various functional nucleic acids into nanoparticles offer advantages for applications in diverse diagnostic and therapeutic approaches. However, due to the novelty of this approach, nucleic acid nanoparticles (NANPs) are not yet used in the clinic. The immune recognition of NANPs is among the areas of preclinical investigation aimed at enabling the translation of these novel materials into clinical settings. NANPs' interactions with the complement system, coagulation systems, and immune cells are essential components of their preclinical safety portfolio. It has been established that NANPs' physicochemical properties-composition, shape, and size-determine their interactions with immune cells (primarily blood plasmacytoid dendritic cells and monocytes), enable recognition by pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs), and mediate the subsequent cytokine response. However, unlike traditional therapeutic nucleic acids (e.g., CpG oligonucleotides), NANPs do not trigger a cytokine response unless they are delivered into the cells using a carrier. Recently, it was discovered that the type of carrier provides an additional tool for regulating both the spectrum and the magnitude of the cytokine response to NANPs. Herein, we review the current knowledge of NANPs' interactions with various components of the immune system to emphasize the unique properties of these nanomaterials and highlight opportunities for their use in vaccines and immunotherapy.

Functionally-interdependent shape-switching nanoparticles with controllable properties.

We introduce a new concept that utilizes cognate nucleic acid nanoparticles which are fully complementary and functionally-interdependent to each other. In the described approach, the physical interaction between sets of designed nanoparticles initiates a rapid isothermal shape change which triggers the activation of multiple functionalities and biological pathways including transcription, energy transfer, functional aptamers and RNA interference. The individual nanoparticles are not active and have controllable kinetics of re-association and fine-tunable chemical and thermodynamic stabilities. Computational algorithms were developed to accurately predict melting temperatures of nanoparticles of various compositions and trace the process of their re-association in silico. Additionally, tunable immunostimulatory properties of described nanoparticles suggest that the particles that do not induce pro-inflammatory cytokines and high levels of interferons can be used as scaffolds to carry therapeutic oligonucleotides, while particles with strong interferon and mild pro-inflammatory cytokine induction may qualify as vaccine adjuvants. The presented concept provides a simple, cost-effective and straightforward model for the development of combinatorial regulation of biological processes in nucleic acid nanotechnology.

Expression of the cellular prion protein affects posttransfusion recovery and survival of red blood cells in mice.

BACKGROUND: Cellular prion protein (PrP(C) ) is expressed on various cell types including red blood cells (RBCs). The PrP(C) plays a key role in the pathogenesis of prion diseases, but its physiologic function remains unclear. PrP(C) is expressed on CD34+ hematopoietic stem cells and its expression is regulated during blood cell differentiation including the erythroid line. STUDY DESIGN AND METHODS: We investigated the role of PrP(C) in RBC survival in circulation by transfusing a mix of biotin-labeled RBCs from wild-type (WT) and PrP knockout (KO) mice to groups of recipient mice (WT and KO). The proportion of biotinylated RBCs in peripheral blood was estimated by flow cytometry. RESULTS: KO RBCs displayed a markedly higher first-day posttransfusion recovery but had a decreased survival in circulation when compared to WT RBCs. Similar results were obtained in all groups of transfused mice, irrespective of RBCs biotinylation level. In addition, we confirmed this finding in an analogous study using Tga20 mice overexpressing PrP(C) and KO mice of a different genetic background. CONCLUSION: Our results demonstrate that PrP(C) expression affects RBC recovery and survival in circulation.

Multifunctional RNA nanoparticles.

Our recent advancements in RNA nanotechnology introduced novel nanoscaffolds (nanorings); however, the potential of their use for biomedical applications was never fully revealed. As presented here, besides functionalization with multiple different short interfering RNAs for combinatorial RNA interference (e.g., against multiple HIV-1 genes), nanorings also allow simultaneous embedment of assorted RNA aptamers, fluorescent dyes, proteins, as well as recently developed RNA-DNA hybrids aimed to conditionally activate multiple split functionalities inside cells.

Dynamic Nanostructures for Conditional Activation and Deactivation of Biological Pathways.

Nucleic acid nanotechnology utilizes natural and synthetic structural motifs to build versatile nucleic acid nanoparticles (NANPs). These rationally designed assemblies can be further equipped with functional nucleic acids and other molecules such as peptides, fluorescent dyes, etc. In addition to nucleic acids that directly interact with the regulated target gene transcripts, NANPs can display decoys, wherein the oligonucleotide stretches with transcription factor binding sequences, preventing transcription initiation. The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a group of five crucial transcription factors regulating the pathogenesis of inflammatory diseases and cancer; as such, they are relevant targets for therapy. One therapeutic approach involves interdependent self-recognizing hybridized DNA/RNA fibers designed to bind NF-κB and prevent its interaction with the promotor region of NF-κB-dependent genes involved in inflammatory responses. Decoying NF-κB results in the inability to initiate transcription of regulated genes, showing a promising approach to gene regulation and gene therapy. The protocol described herein provides detailed steps for the synthesis of NF-κB decoy fibers, as well as their characterization using polyacrylamide gel electrophoresis (to confirm desired physicochemical properties and purity) and functional bioassays (to confirm desired biological activity).

Therapeutic immunomodulation by rationally designed nucleic acids and nucleic acid nanoparticles.

The immune system has evolved to defend organisms against exogenous threats such as viruses, bacteria, fungi, and parasites by distinguishing between "self" and "non-self". In addition, it guards us against other diseases, such as cancer, by detecting and responding to transformed and senescent cells. However, for survival and propagation, the altered cells and invading pathogens often employ a wide range of mechanisms to avoid, inhibit, or manipulate the immunorecognition. As such, the development of new modes of therapeutic intervention to augment protective and prevent harmful immune responses is desirable. Nucleic acids are biopolymers essential for all forms of life and, therefore, delineating the complex defensive mechanisms developed against non-self nucleic acids can offer an exciting avenue for future biomedicine. Nucleic acid technologies have already established numerous approaches in therapy and biotechnology; recently, rationally designed nucleic acids nanoparticles (NANPs) with regulated physiochemical properties and biological activities has expanded our repertoire of therapeutic options. When compared to conventional therapeutic nucleic acids (TNAs), NANP technologies can be rendered more beneficial for synchronized delivery of multiple TNAs with defined stabilities, immunological profiles, and therapeutic functions. This review highlights several recent advances and possible future directions of TNA and NANP technologies that are under development for controlled immunomodulation.

Underestimation of the expression of cellular prion protein on human red blood cells.

BACKGROUND: Recent transmissions of variant Creutzfeldt-Jakob disease by blood transfusion emphasize the need for the development of prion screening tests. The detection of prions in blood is complicated by the presence of poorly characterized cellular prion protein (PrP(C) ) in both plasma and blood cells. According to published studies, most of PrP(C) in blood cells resides in platelets (PLTs) and white blood cells. STUDY DESIGN AND METHODS: To clarify conflicting reports about the quantity of PrP(C) associated with human red blood cells (RBCs), quantitative flow cytometry, Western blot (WB), and enzyme-linked immunosorbent assay (ELISA) were used to measure protein levels in healthy donors. RESULTS: RBCs expressed 290 ± 140 molecules of PrP(C) per cell, assuming equimolar binding of monoclonal antibody (MoAb) 6H4 to PrP(C). Binding of alternate PrP(C) MoAbs, FH11 and 3F4, was substantially lower. WB estimated the level of PrP(C) per cell on RBCs to be just four times lower than in PLTs. A similar level of PrP(C) was detected using ELISA. The weak binding of commonly used MoAb 3F4 was not caused by PrP(C) conformation, truncation, or glycosylation, suggesting a covalent modification, likely glycation, of the 3F4 epitope. CONCLUSIONS: Taken together, human RBCs express low but significant amounts of PrP(C) /cell, which makes them, due to high RBC numbers, major contributors to the pool of cell-associated PrP(C) in blood. Previous reports utilizing MoAb 3F4 may have underestimated the amount of PrP(C) in RBCs. Likewise, screening tests for the presence of the abnormal prion protein in blood may be difficult if the abnormal protein is modified similar to RBC PrP(C).

Development of monoclonal antibodies specific for glycated prion protein.

Transmissive spongiform encephalopathies (TSE) are neurodegenerative diseases characterized by depositions of abnormally folded prion protein (PrP(TSE)) in brain. PrP(TSE) is at present the only specific biochemical marker of human and animal TSE. As deposits of PrP(TSE) remain in the body for long periods, there is substantial chance of them being nonenzymatically modified by glycation. The detection of glycated PrP(TSE) may have potential to serve as a diagnostic marker. Monoclonal antibodies specific for carboxymethyl lysine/arginine-modified prion protein were prepared. Recombinant human prion protein (rhPrP) was bacterially expressed and purified by affinity chromatography. rhPrP was modified by glyoxylic acid that introduces carboxymethyl groups on lysine and arginine residues present within the molecule of the protein. Modified rhPrP (rhPrP-CML) was used for immunization of 6 mice, and 960 hybridoma cells were prepared. Screening of cell supernatants resulted in the selection of four promising clones. One of them (EM-31) strongly reacts with human and mouse recombinant PrP-CML, and three other clones react also with CML in vitro modified human and mouse brain PrP. Besides possible implication in TSE diagnostics, the antibodies may serve as tolls to advance our knowledge regarding the role of glycation in the prion pathophysiology.

Controlled Organization of Inorganic Materials Using Biological Molecules for Activating Therapeutic Functionalities.

Precise control over the assembly of biocompatible three-dimensional (3D) nanostructures would allow for programmed interactions within the cellular environment. Nucleic acids can be used as programmable crosslinkers to direct the assembly of quantum dots (QDs) and tuned to demonstrate different interparticle binding strategies. Morphologies of self-assembled QDs are evaluated via gel electrophoresis, transmission electron microscopy, small-angle X-ray scattering, and dissipative particle dynamics simulations, with all results being in good agreement. The controlled assembly of 3D QD organizations is demonstrated in cells via the colocalized emission of multiple assembled QDs, and their immunorecognition is assessed via enzyme-linked immunosorbent assays. RNA interference inducers are also embedded into the interparticle binding strategy to be released in human cells only upon QD assembly, which is demonstrated by specific gene silencing. The programmability and intracellular activity of QD assemblies offer a strategy for nucleic acids to imbue the structure and therapeutic function into the formation of complex networks of nanostructures, while the photoluminescent properties of the material allow for optical tracking in cells in vitro.

Challenges to optimizing RNA nanostructures for large scale production and controlled therapeutic properties.

Nucleic acids have been utilized to construct an expansive collection of nanoarchitectures varying in design, physicochemical properties, cellular processing and biomedical applications. However, the broader therapeutic adaptation of nucleic acid nanoassemblies in general, and RNA-based nanoparticles in particular, have faced several challenges in moving towards (pre)clinical settings. For one, the large-batch synthesis of nucleic acids is still under development, with multi-stranded and chemically modified assemblies requiring greater production capacity while maintaining consistent medical-grade outputs. Furthermore, the unknown immunostimulation by these nanomaterials poses additional challenges, necessary to be overcome for optimizing future development of clinically approved RNA nanoparticles.

Innate immune responses triggered by nucleic acids inspire the design of immunomodulatory nucleic acid nanoparticles (NANPs).

The unknown immune stimulation by nucleic acid nanoparticles (NANPs) has become one of the major impediments to a broad spectrum of clinical developments of this novel technology. Having evolved to defend against bacterial and viral nucleic acids, mammalian cells have established patterns of recognition that are also the pathways through which NANPs can be processed. Explorations into the immune stimulation brought about by a vast diversity of known NANPs have shown that variations in design correlate with variations in immune response. Therefore, as the mechanisms of stimulation are further elucidated, these trends are now being taken into account in the design phase to allow for development of NANPs that are tailored for controlled immune activation or quiescence.

Aptamers as Modular Components of Therapeutic Nucleic Acid Nanotechnology.

Nucleic acids play a central role in all domains of life, either as genetic blueprints or as regulators of various biochemical pathways. The chemical makeup of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), generally represented by a sequence of four monomers, also provides precise instructions for folding and higher-order assembly of these biopolymers that, in turn, dictate biological functions. The sequence-based specific 3D structures of nucleic acids led to the development of the directed evolution of oligonucleotides, SELEX (systematic evolution of ligands by exponential enrichment), against a chosen target molecule. Among the variety of functions, selected oligonucleotides named aptamers also allow targeting of cell-specific receptors with antibody-like precision and can deliver functional RNAs without a transfection agent. The advancements in the field of customizable nucleic acid nanoparticles (NANPs) opened avenues for the design of nanoassemblies utilizing aptamers for triggering or blocking cell signaling pathways or using aptamer-receptor combinations to activate therapeutic functionalities. A recent selection of fluorescent aptamers enables real-time tracking of NANP formation and interactions. The aptamers are anticipated to contribute to the future development of technologies, enabling an efficient assembly of functional NANPs in mammalian cells or in vivo. These research topics are of top importance for the field of therapeutic nucleic acid nanotechnology with the promises to scale up mass production of NANPs suitable for biomedical applications, to control the intracellular organization of biological materials to enhance the efficiency of biochemical pathways, and to enhance the therapeutic potential of NANP-based therapeutics while minimizing undesired side effects and toxicities.

Expression of prion protein in mouse erythroid progenitors and differentiating murine erythroleukemia cells.

Prion diseases have been observed to deregulate the transcription of erythroid genes, and prion protein knockout mice have demonstrated a diminished response to experimental anemia. To investigate the role of the cellular prion protein (PrP(C)) in erythropoiesis, we studied the protein's expression on mouse erythroid precursors in vivo and utilized an in vitro model of the erythroid differentiation of murine erythroleukemia cells (MEL) to evaluate the effect of silencing PrP(C) through RNA interference.The expression of PrP(C) and selected differentiation markers was analyzed by quantitative multicolor flow cytometry, western blot analysis and quantitative RT-PCR. The silencing of PrP(C) expression in MEL cells was achieved by expression of shRNAmir from an integrated retroviral vector genome. The initial upregulation of PrP(C) expression in differentiating erythroid precursors was detected both in vivo and in vitro, suggesting PrP(C)'s importance to the early stages of differentiation. The upregulation was highest on early erythroblasts (16200±3700 PrP(C) / cell) and was followed by the gradual decrease of PrP(C) level with the precursor's maturation reaching 470±230 PrP(C) / cell on most mature CD71(-)Ter119(+) small precursors. Interestingly, the downregulation of PrP(C) protein with maturation of MEL cells was not accompanied by the decrease of PrP mRNA. The stable expression of anti-Prnp shRNAmir in MEL cells led to the efficient (>80%) silencing of PrP(C) levels. Cell growth, viability, hemoglobin production and the transcription of selected differentiation markers were not affected by the downregulation of PrP(C).In conclusion, the regulation of PrP(C) expression in differentiating MEL cells mimics the pattern detected on mouse erythroid precursors in vivo. Decrease of PrP(C) protein expression during MEL cell maturation is not regulated on transcriptional level. The efficient silencing of PrP(C) levels, despite not affecting MEL cell differentiation, enables created MEL lines to be used for studies of PrP(C) cellular function.