Enhancing Immunomodulation on Innate Immunity by Shape Transition Among RNA Triangle, Square and Pentagon Nanovehicles

Modulation of immune response is important in cancer immunotherapy, vaccine adjuvant development and inflammatory or immune disease therapy. Here we report the development of new immunomodulators via control of shape transition among RNA triangle, square and pentagon. Changing one RNA strand in polygons automatically induced the stretching of the interior angle from 60° to 90° or 108°, resulting in self-assembly of elegant RNA triangles, squares and pentagons. When immunological adjuvants were incorporated, their immunomodulation effect for cytokine TNF-α and IL-6 induction was greatly enhanced in vitro and in animals up to 100-fold, while RNA polygon controls induced unnoticeable effect. The RNA nanoparticles were delivered to macrophages specifically. The degree of immunostimulation greatly depended on the size, shape and number of the payload per nanoparticles. Stronger immune response was observed when the number of adjuvants per polygon was increased, demonstrating the advantage of shape transition from triangle to pentagon

βグルカン光学と超分子光学を用いたワクチンナノメディシンの新規開発

本研究は、多糖と核酸医薬が形成する複合体を用いたがんワクチン開発研究である。特に、複合体のサイズ効果に焦点を当てたアジュバントデリバリーの研究をしている。核酸のハイブリダイゼーションからなる架橋点を導入することで細胞取り込みに適したサイズを検討した。がんワクチンモデルにおいて、高い免疫活性と抗腫瘍効果を示し、がんワクチンのアジュバントとして有効であることを明らかにした。北九州市立大

RNA-Based Compositions and Adjuvants for Prophylactic and Therapeutic Treatment

The present invention is directed towards an artificial RNA nano structure comprising multiple external strands of RNA, each external strand comprising about 40-50 nucleotides; one internal strand of RNA comprising more than about 50 nucleotides; the internal strands and external strands assembled to form a triangle nanostructure, a square nanostructure, or a polygon nanostructure and a pRNA three-way junction (3WJ) motif at each vertex of the nanostructure. Such nanostructure can be provided in a composition together with an adjuvant for use in inducing the production of high affinity neutralizing antibodies or inhibitory antibodies, inducing the production of cytokines, inducing an immune response in a subject, or a combination thereof

RNA AS A UNIQUE POLYMER TO BUILD CONTROLLABLE NANOSTRUCTURES FOR NANOMEDICINE AND NANOTECHNOLOGY

RNA nanotechnology is an emerging field that involves the design, construction and functionalization of nanostructures composed mainly of RNA for applications in biomedical and material sciences. RNA is a unique polymer with structural simplicity like DNA and functional diversity like proteins. A variety of RNA nanostructures have been reported with different geometrical structures and functionalities. This dissertation describes the design and construction of novel two-dimensional and three-dimensional self-assembled RNA nanostructures with applications in therapeutics delivery, cancer targeting and immunomodulation. Firstly, by using the ultra-stable pRNA three-way junction motif with controllable angles and arm lengths, tetrahedral architectures composed purely of RNA were successfully assembled via one-pot bottom-up assembly with high efficiency and thermal stability. By introducing arm sizes of 22 bp and 55 bp, two RNA tetrahedrons with similar global contour structure but with different sizes of 8 nm and 17 nm were successfully assembled. The RNA tetrahedrons were also highly amenable to functionalization. Fluorogenic RNA aptamers, ribozyme, siRNA, and protein-binding RNA aptamers were integrated into the tetrahedrons by simply fusing the respective sequences with the tetrahedral core modules. Secondly, I reported the design and construction of molecularly defined RNA cages with cube and dodecahedron shapes based on the stable pRNA 3WJ. The RNA cages can be easily self-assembled by single-step annealing. The RNA cages were further characterized by gel electrophoresis, cryo-electron microscopy and atomic force microscopy, confirming the spontaneous formation of the RNA cages. I also demonstrated that the constructed RNA cages could be used to deliver model drugs such as immunomodulatory CpG DNA into cells and elicit enhanced immune responses. Thirdly, by using the modular multi-domain strategy, molecular defined RNA nanowires can be successfully self-assembled via a bottom-up approach. Only four different 44-nucleotide single-stranded RNAs were used to assemble the RNA nanowire. The reported RNA nanowire has the potential to be explored in the future as the carrier for drug delivery or matrix for tissue engineering. Fourthly, the construction of RNA polygons for delivering immunoactive CpG oligonucleotides will be presented. When CpG oligonucleotides were incorporated into the RNA polygons, their immunomodulation effect for cytokine TNF-α and IL-6 induction was greatly enhanced, while RNA polygon controls induced unnoticeable cytokine induction. Moreover, the RNA polygons were delivered to macrophages specifically and the degree of immunostimulation greatly depended on the size, shape, and the number of payload per RNA polygon. Collectively, these findings demonstrated RNA nanotechnology can produce controllable nanostructures with different functionalities and result in potential applications in nanomedicine and nanotechnology

Synthetic Nanoparticles for Vaccines and Immunotherapy

The immune system plays a critical role in our health. No other component of human physiology plays a decisive role in as diverse an array of maladies, from deadly diseases with which we are all familiar to equally terrible esoteric conditions: HIV, malaria, pneumococcal and influenza infections; cancer; atherosclerosis; autoimmune diseases such as lupus, diabetes, and multiple sclerosis. The importance of understanding the function of the immune system and learning how to modulate immunity to protect against or treat disease thus cannot be overstated. Fortunately, we are entering an exciting era where the science of immunology is defining pathways for the rational manipulation of the immune system at the cellular and molecular level, and this understanding is leading to dramatic advances in the clinic that are transforming the future of medicine.1,2 These initial advances are being made primarily through biologic drugs– recombinant proteins (especially antibodies) or patient-derived cell therapies– but exciting data from preclinical studies suggest that a marriage of approaches based in biotechnology with the materials science and chemistry of nanomaterials, especially nanoparticles, could enable more effective and safer immune engineering strategies. This review will examine these nanoparticle-based strategies to immune modulation in detail, and discuss the promise and outstanding challenges facing the field of immune engineering from a chemical biology/materials engineering perspectiveNational Institutes of Health (U.S.) (Grants AI111860, CA174795, CA172164, AI091693, and AI095109)United States. Department of Defense (W911NF-13-D-0001 and Awards W911NF-07-D-0004

Efficient delivery of immunostimulatory DNA to mouse and human immune cells through the construction of polypod-like structured DNA.

Investigation of mouse macrophage-like RAW264.7 cells showed that the immunostimulatory activity of CpG DNA is increased by formation of polypod-like structured DNA (polypodna), an assembly consisting of three or more oligodeoxynucleotides. To apply CpG polypodna to immunotherapy, its activity was examined in murine dendritic DC2.4 cells, splenic macrophages, and bone marrow-derived dendritic cells (BMDCs). In all cell types, increasing the pod number increased the cellular uptake of DNA and cytokine release. No significant release of cytokines was observed in macrophages lacking Toll-like receptor 9. Similar results were obtained after intradermal injection of polypodna. The polypodna preparations produced significantly higher amounts of interferon α in human peripheral blood mononuclear cells (PBMCs) compared with single-stranded DNA. The conditioned medium of hexapodna-treated human PBMCs effectively inhibited the activity of a hepatitis C virus subgenomic replicon reporter system. These results indicate that polypodna preparations are useful as an immunostimulator