A Piecewise Design Approach to Engineering a Miniature ACE2 Mimic to Bind SARS-CoV-2.

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues its global spread, the exploration of novel therapeutic and diagnostic strategies is still needed. The virus enters host cells by binding the angiotensin-converting enzyme 2 (ACE2) receptor through the spike protein. Here, we develop an engineered, small, stable, and catalytically inactive version of ACE2, termed miniature ACE2 (mACE2), designed to bind the spike protein with high affinity. Employing a magnetic nanoparticle-based assay, we harnessed the strong binding affinity of mACE2 to develop a sensitive and specific platform for the detection or neutralization of SARS-CoV-2. Our findings highlight the potential of engineered mACE2 as a valuable tool in the fight against SARS-CoV-2. The success of developing such a small reagent based on a piecewise molecular design serves as a proof-of-concept approach for the rapid deployment of such agents to diagnose and fight other viral diseases.

Nanoscale metal-organic frameworks for the delivery of nucleic acids to cancer cells.

Therapeutic nucleic acids (TNAs) are gaining increasing interest in the treatment of severe diseases including viral infections, inherited disorders, and cancers. However, the efficacy of intracellularly functioning TNAs is also reliant upon their delivery into the cellular environment, as unmodified nucleic acids are unable to cross the cell membrane mainly due to charge repulsion. Here we show that TNAs can be effectively delivered into the cellular environment using engineered nanoscale metal-organic frameworks (nanoMOFs), with the additional ability to tailor which cells receive the therapeutic cargo determined by the functional moieties grafted onto the nanoMOF's surface. This study paves the way to integrate the highly ordered programmable nucleic acids into larger-scale functionalized nanoassemblies.

Artificial Immune Cell, AI-cell, a New Tool to Predict Interferon Production by Peripheral Blood Monocytes in Response to Nucleic Acid Nanoparticles.

Nucleic acid nanoparticles, or NANPs, rationally designed to communicate with the human immune system, can offer innovative therapeutic strategies to overcome the limitations of traditional nucleic acid therapies. Each set of NANPs is unique in their architectural parameters and physicochemical properties, which together with the type of delivery vehicles determine the kind and the magnitude of their immune response. Currently, there are no predictive tools that would reliably guide the design of NANPs to the desired immunological outcome, a step crucial for the success of personalized therapies. Through a systematic approach investigating physicochemical and immunological profiles of a comprehensive panel of various NANPs, the research team developes and experimentally validates a computational model based on the transformer architecture able to predict the immune activities of NANPs. It is anticipated that the freely accessible computational tool that is called an "artificial immune cell," or AI-cell, will aid in addressing the current critical public health challenges related to safety criteria of nucleic acid therapies in a timely manner and promote the development of novel biomedical tools.

The Application of Light-Assisted Drying to the Thermal Stabilization of Nucleic Acid Nanoparticles.

Background: Cold-chain storage can be challenging and expensive for the transportation and storage of biologics, especially in low-resource settings. Nucleic acid nanoparticles (NANPs) are an example of new biological products that require refrigerated storage. Light-assisted drying (LAD) is a new processing technique to prepare biologics for anhydrous storage in a trehalose amorphous solid matrix at ambient temperatures. In this study, LAD was used to thermally stabilize four types of NANPs with differing structures and melting temperatures. Methods: Small volume samples (10 µL) containing NANPs were irradiated with a 1064 nm laser to speed the evaporation of water and create an amorphous trehalose preservation matrix. Samples were then stored for 1 month at 4°C or 20°C. A FLIR C655 mid-IR camera was used to record the temperature of samples during processing. The trehalose matrix was characterized using polarized light imaging (PLI) to determine if crystallization occurred during processing or storage. Damage to LAD-processed NANPs was assessed after processing and storage using gel electrophoresis. Results: Based on the end moisture content (EMC) as a function time and the thermal histories of samples, a LAD processing time of 30 min is sufficient to achieve low EMCs for the 10 µL samples used in this study. PLI demonstrates that the trehalose matrix was resistant to crystallization during processing and after storage at 4°C and at room temperature. The native-polyacrylamide gel electrophoresis results for DNA cubes, RNA cubes, and RNA rings indicate that the main structures of these NANPs were not damaged significantly after LAD processing and being stored at 4°C or at room temperature for 1 month. Conclusions: These preliminary studies indicate that LAD processing can stabilize NANPs for dry-state storage at room temperature, providing an alternative to refrigerated storage for these nanomedicine products.

Locking and Unlocking Thrombin Function Using Immunoquiescent Nucleic Acid Nanoparticles with Regulated Retention In Vivo.

The unbalanced coagulation of blood is a life-threatening event that requires accurate and timely treatment. We introduce a user-friendly biomolecular platform based on modular RNA-DNA anticoagulant fibers programmed for reversible extracellular communication with thrombin and subsequent control of anticoagulation via a "kill-switch" mechanism that restores hemostasis. To demonstrate the potential of this reconfigurable technology, we designed and tested a set of anticoagulant fibers that carry different thrombin-binding aptamers. All fibers are immunoquiescent, as confirmed in freshly collected human peripheral blood mononuclear cells. To assess interindividual variability, the anticoagulation is confirmed in the blood of human donors from the U.S. and Brazil. The anticoagulant fibers reveal superior anticoagulant activity and prolonged renal clearance in vivo in comparison to free aptamers. Finally, we confirm the efficacy of the "kill-switch" mechanism in vivo in murine and porcine models.

Adaptation-proof SARS-CoV-2 vaccine design.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surface spike glycoprotein - a major antibody target - is critical for virus entry via engagement of human angiotensin-converting enzyme 2 (ACE2) receptor. Despite successes with existing vaccines and therapies that primarily target the receptor binding domain (RBD) of the spike protein, the susceptibility of RBD to mutations provides escape routes for the SARS-CoV-2 from neutralizing antibodies. On the other hand, structural conservation in the spike protein can be targeted to reduce escape mutations and achieve broad protection. Here, we designed candidate stable immunogens that mimic surface features of selected conserved regions of spike protein through 'epitope grafting,' in which we present the target epitope topology on diverse heterologous scaffolds that can structurally accommodate the spike epitopes. Structural characterization of the epitope-scaffolds showed stark agreement with our computational models and target epitopes. The sera from mice immunized with engineered designs display epitope-scaffolds and spike binding activity. We also demonstrated the utility of the designed epitope-scaffolds in diagnostic applications. Taken all together, our study provides important methodology for targeting the conserved, non-RBD structural motifs of spike protein for SARS-CoV-2 epitope vaccine design and demonstrates the potential utility of 'epitope grafting' in rational vaccine design.

Discrimination of RNA fiber structures using solid-state nanopores.

RNA fibers are a class of biomaterials that can be assembled using HIV-like kissing loop interactions. Because of the programmability of molecular design and low immunorecognition, these structures present an interesting opportunity to solve problems in nanobiotechnology and synthetic biology. However, the experimental tools to fully characterize and discriminate among different fiber structures in solution are limited. Herein, we utilize solid-state nanopore experiments and Brownian dynamics simulations to characterize and distinguish several RNA fiber structures that differ in their degrees of branching. We found that, regardless of the electrolyte type and concentration, fiber structures that have more branches produce longer and deeper ionic current blockades in comparison to the unbranched fibers. Experiments carried out at temperatures ranging from 20-60 °C revealed almost identical distributions of current blockade amplitudes, suggesting that the kissing loop interactions in fibers are resistant to heating within this range.

Anhydrous Nucleic Acid Nanoparticles for Storage and Handling at Broad Range of Temperatures.

Recent advances in nanotechnology now allow for the methodical implementation of therapeutic nucleic acids (TNAs) into modular nucleic acid nanoparticles (NANPs) with tunable physicochemical properties which can match the desired biological effects, provide uniformity, and regulate the delivery of multiple TNAs for combinatorial therapy. Despite the potential of novel NANPs, the maintenance of their structural integrity during storage and shipping remains a vital issue that impedes their broader applications. Cold chain storage is required to maintain the potency of NANPs in the liquid phase, which greatly increases transportation costs. To promote long-term storage and retention of biological activities at higher temperatures (e.g., +50 °C), a panel of representative NANPs is first exposed to three different drying mechanisms-vacuum concentration (SpeedVac), lyophilization (Lyo), and light-assisted drying (LAD)-and then rehydrated and analyzed. While SpeedVac primarily operates using heat, Lyo avoids temperature increases by taking advantage of pressure reduction and LAD involves a near-infrared laser for uniform drying in the presence of trehalose. This work compares and defines refinements crucial in formulating an optimal strategy for producing stable, fully functional NANPs and presents a forward advancement in their development for clinical applications.

Adaptation-proof SARS-CoV-2 vaccine design.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surface spike glycoprotein - a major antibody target - is critical for virus entry via engagement of human angiotensin-converting enzyme 2 (ACE2) receptor. Despite successes with existing vaccines and therapies that primarily target the receptor binding domain (RBD) of the spike protein, the susceptibility of RBD to mutations provides escape routes for the SARS-CoV-2 from neutralizing antibodies. On the other hand, structural conservation in the spike protein can be targeted to reduce escape mutations and achieve broad protection. Here, we designed candidate stable immunogens that mimic surface features of selected conserved regions of spike protein through 'epitope grafting,' in which we present the target epitope topology on diverse heterologous scaffolds that can structurally accommodate the spike epitopes. Structural characterization of the epitope-scaffolds showed stark agreement with our computational models and target epitopes. The sera from mice immunized with engineered designs display epitope-scaffolds and spike binding activity. We also demonstrated the utility of the designed epitope-scaffolds in diagnostic applications. Taken all together, our study provides important methodology for targeting the conserved, non-RBD structural motifs of spike protein for SARS-CoV-2 epitope vaccine design and demonstrates the potential utility of 'epitope grafting' in rational vaccine design.

Expanding Structural Space for Immunomodulatory Nucleic Acid Nanoparticles (Nanps) via Spatial Arrangement of Their Therapeutic Moieties.

Different therapeutic nucleic acids (TNAs) can be unified in a single structure by their elongation with short oligonucleotides designed to self-assemble into nucleic acid nanoparticles (NANPs). With this approach, therapeutic cocktails with precisely controlled composition and stoichiometry of active ingredients can be delivered to the same diseased cells for enhancing pharmaceutical action. In this work, an additional nanotechnology-based therapeutic option that enlists a biocompatible NANP-encoded platform for their controlled patient-specific immunorecognition is explored. For this, a set of representative functional NANPs is extensively characterized in vitro, ex vivo, and in vivo and then further analyzed for immunostimulation of human peripheral blood mononuclear cells freshly collected from healthy donor volunteers. The results of the study present the advancement of the current TNA approach toward personalized medicine and offer a new strategy to potentially address top public health challenges related to drug overdose and safety through the biodegradable nature of the functional platform with immunostimulatory regulation.

The International Society of RNA Nanotechnology and Nanomedicine (ISRNN): The Present and Future of the Burgeoning Field.

The International Society of RNA Nanotechnology and Nanomedicine (ISRNN) hosts an annual meeting series focused on presenting the latest research achievements involving RNA-based therapeutics and strategies, aiming to expand their current biomedical applications while overcoming the remaining challenges of the burgeoning field of RNA nanotechnology. The most recent online meeting hosted a series of engaging talks and discussions from an international cohort of leading nanotechnologists that focused on RNA modifications and modulation, dynamic RNA structures, overcoming delivery limitations using a variety of innovative platforms and approaches, and addressing the newly explored potential for immunomodulation with programmable nucleic acid nanoparticles. In this Nano Focus, we summarize the main discussion points, conclusions, and future directions identified during this two-day webinar as well as more recent advances to highlight and to accelerate this exciting field.

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.

Programmable DNA-augmented hydrogels for controlled activation of human lymphocytes.

Contractile forces within the planar interface between T cell and antigen-presenting surface mechanically stimulate T cell receptors (TCR) in the mature immune synapses. However, the origin of mechanical stimulation during the initial, i.e., presynaptic, microvilli-based TCR activation in the course of immune surveillance remains unknown and new tools to help address this problem are needed. In this work, we develop nucleic acid nanoassembly (NAN)-based technology for functionalization of hydrogels using isothermal toehold-mediated reassociation of RNA/DNA heteroduplexes. Resulting platform allows for regulation with NAN linkers of 3D force momentum along the TCR mechanical axis, whereas hydrogels contribute to modulation of 2D shear modulus. By utilizing different lengths of NAN linkers conjugated to polyacrylamide gels of different shear moduli, we demonstrate an efficient capture of human T lymphocytes and tunable activation of TCR, as confirmed by T-cell spreading and pY foci.

DNA-Templated Fluorescent Silver Nanoclusters Inhibit Bacterial Growth While Being Non-Toxic to Mammalian Cells.

Silver has a long history of antibacterial effectiveness. The combination of atomically precise metal nanoclusters with the field of nucleic acid nanotechnology has given rise to DNA-templated silver nanoclusters (DNA-AgNCs) which can be engineered with reproducible and unique fluorescent properties and antibacterial activity. Furthermore, cytosine-rich single-stranded DNA oligonucleotides designed to fold into hairpin structures improve the stability of AgNCs and additionally modulate their antibacterial properties and the quality of observed fluorescent signals. In this work, we characterize the sequence-specific fluorescence and composition of four representative DNA-AgNCs, compare their corresponding antibacterial effectiveness at different pH, and assess cytotoxicity to several mammalian cell lines.

Simultaneous silencing of lysophosphatidylcholine acyltransferases 1-4 by nucleic acid nanoparticles (NANPs) improves radiation response of melanoma cells.

Radiation induces the generation of platelet-activating factor receptor (PAF-R) ligands, including PAF and oxidized phospholipids. Alternatively, PAF is also synthesized by the biosynthetic enzymes lysophosphatidylcholine acyltransferases (LPCATs) which are expressed by tumor cells including melanoma. The activation of PAF-R by PAF and oxidized lipids triggers a survival response protecting tumor cells from radiation-induced cell death, suggesting the involvement of the PAF/PAF-R axis in radioresistance. Here, we investigated the role of LPCATs in the melanoma cell radiotherapy response. LPCAT is a family of four enzymes, LPCAT1-4, and modular nucleic acid nanoparticles (NANPs) allowed for the simultaneous silencing of all four LPCATs. We found that the in vitro simultaneous silencing of all four LPCAT transcripts by NANPs enhanced the therapeutic effects of radiation in melanoma cells by increasing cell death, reducing long-term cell survival, and activating apoptosis. Thus, we propose that NANPs are an effective strategy for improving radiotherapy efficacy in melanomas.

Nucleic acid nanoparticles (NANPs) as molecular tools to direct desirable and avoid undesirable immunological effects.

Nucleic acid nanoparticles (NANPs) represent a highly versatile molecular platform for the targeted delivery of various therapeutics. However, despite their promise, further clinical translation of this innovative technology can be hindered by immunological off-target effects. All human cells are equipped with an arsenal of receptors that recognize molecular patterns specific to foreign nucleic acids and understanding the rules that guide this recognition offer the key rationale for the development of therapeutic NANPs with tunable immune stimulation. Numerous recent studies have provided increasing evidence that in addition to NANPs' physicochemical properties and therapeutic effects, their interactions with cells of the immune system can be regulated through multiple independently programmable architectural parameters. The results further suggest that defined immunomodulation by NANPs can either support their immunoquiescent delivery or be used for conditional stimulation of beneficial immunological responses.

Induction of Cytokines by Nucleic Acid Nanoparticles (NANPs) Depends on the Type of Delivery Carrier.

Recent insights into the immunostimulatory properties of nucleic acid nanoparticles (NANPs) have demonstrated that variations in the shape, size, and composition lead to distinct patterns in their immunostimulatory properties. While most of these studies have used a single lipid-based carrier to allow for NANPs' intracellular delivery, it is now apparent that the platform for delivery, which has historically been a hurdle for therapeutic nucleic acids, is an additional means to tailoring NANP immunorecognition. Here, the use of dendrimers for the delivery of NANPs is compared to the lipid-based platform and the differences in resulting cytokine induction are presented.

Combination of Nucleic Acid and Mesoporous Silica Nanoparticles: Optimization and Therapeutic Performance In Vitro.

Programmable nucleic acid nanoparticles (NANPs) with precisely controlled functional compositions can regulate the conditional activation of various biological pathways and responses in human cells. However, the intracellular delivery of NANPs alone is hindered by their susceptibility to nuclease activity and inefficient crossing of biological membranes. In this work, we optimized the internalization and therapeutic performance of several representative NANPs delivered with mesoporous silica nanoparticles (MSNPs) tailored for efficient electrostatic association with NANPs. We compared the immunostimulatory properties of different NA-MS-NP complexes formed with globular, planar, and fibrous NANPs and demonstrated the maximum immunostimulation for globular NANPs. As a proof of concept, we assessed the specific gene silencing by NA-MS-NP complexes functionalized with siRNA targeting green fluorescent protein expressed in triple-negative human breast cancer cells. We showed that the fibrous NANPs have the highest silencing efficiency when compared to globular or planar counterparts. Finally, we confirmed the multimodal ability of MSNPs to co-deliver a chemotherapy drug, doxorubicin, and NANPs targeting apoptosis regulator gene BCL2 in triple-negative breast cancer and melanoma cell lines. Overall, the combination of NANPs and MSNPs may become a new promising approach to efficiently treat cancer and other diseases via the simultaneous targeting of various pathways.

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.

A cationic amphiphilic co-polymer as a carrier of nucleic acid nanoparticles (Nanps) for controlled gene silencing, immunostimulation, and biodistribution.

Programmable nucleic acid nanoparticles (NANPs) provide controlled coordination of therapeutic nucleic acids (TNAs) and other biological functionalities. Beyond multivalence, recent reports demonstrate that NANP technology can also elicit a specific immune response, adding another layer of customizability to this innovative approach. While the delivery of nucleic acids remains a challenge, new carriers are introduced and tested continuously. Polymeric platforms have proven to be efficient in shielding nucleic acid cargos from nuclease degradation while promoting their delivery and intracellular release. Here, we venture beyond the delivery of conventional TNAs and combine the stable cationic poly-(lactide-co-glycolide)-graft-polyethylenimine with functionalized NANPs. Furthermore, we compare several representative NANPs to assess how their overall structures influence their delivery with the same carrier. An extensive study of various formulations both in vitro and in vivo reveals differences in their immunostimulatory activity, gene silencing efficiency, and biodistribution, with fibrous NANPs advancing for TNA delivery.