Naoto Yoshinaga

Efficient nucleic acid delivery across diverse plants remains challenging because carrier performance often depends on plant species and tissue type. Herein, we developed peptide-based messenger RNA (mRNA)-loaded polyplex micelles decorated with phenylboronic acid (PBA) ligands designed based on the conserved boric acid uptake system in the roots of vascular plants. Among the tested PBA ligands, installation of 3-methyl-substituted PBA possessing a relatively high acid dissociation constant significantly increased cellular mRNA uptake and reporter gene expression in the root tissues of Arabidopsis thaliana , Oryza sativa and Nicotiana benthamiana , revealing the applicability of the same ligand design to both monocot and eudicot species. The PBA ligand effect was pH dependent. All the tested PBA derivatives enhanced mRNA uptake under acidic conditions, where PBA derivatives predominantly adopt uncharged trigonal structures, whereas no enhancement was observed under alkaline conditions, where negatively charged tetrahedral forms predominate. Furthermore, reduced boric acid availability in the culture medium increased the expression of the boric acid channel (NOD26-like intrinsic protein 5;1, NIP5;1), accompanying increased cellular uptake. These findings reveal the substituent structure and ionization state of PBA ligands as important parameters for peptide-based nucleic acid carriers and provide a strategy for broadening transient mRNA-based expression technologies across vascular plants.

Mitochondrial transplantation has gathered much attention as therapeutics to improve multiple mitochondrial functions simultaneously. While the administration of naked mitochondria into the target tissue has demonstrated therapeutic outcomes sufficient to advance to clinical trials, there remain many limitations, including a low cellular uptake efficiency in the target tissue and dysfunction of the isolated mitochondria. To address these issues, engineering approaches have been developed to functionalize the isolated mitochondria. In this review, we focus on the three critical topics for efficient mitochondrial transplantation and outline emerging design rules and their limitations for each purpose: (i) tissue targeting, (ii) protection of mitochondria from external stresses, and (iii) improvement of cellular uptake efficiency. From these achievements, we also discuss the current limitations of mitochondrial transplantation and propose the future direction of the attractive therapeutic methodology.

Mitochondria are essential for cellular functions, such as energy production. Human mitochondrial DNA (mtDNA), encoding 13 distinct genes, two rRNA, and 22 tRNA, is crucial for maintaining vital functions, along with nuclear-encoded mitochondrial proteins. However, mtDNA is prone to somatic mutations due to replication errors and reactive oxygen species exposure. These mutations can accumulate, leading to heteroplasmic conditions associated with severe metabolic diseases. Therefore, developing methodologies to improve mitochondrial health is highly demanded. Introducing nucleic acids directly into mitochondria is a promising strategy to control mitochondrial gene expression. Messenger RNA (mRNA) delivery especially offers several advantages such as faster gene expression and reduced risk of genome integration if accidentally delivered to the cell nucleus. In this study, we investigated the effect of the poly(A) tail length of mRNA on the mitochondrial translation to achieve efficient expression. We used a peptide-based mitochondrial targeting system, mitoNEET-(RH)(9), comprising a mitochondria-targeting sequence (MTS) and a cationic sequence, to deliver mRNA with various poly(A) tails into the mitochondria. The poly(A) tail length significantly affected translational efficiency, with a medium length of 60 nucleotides maximizing protein expression in various cell lines due to enhanced interaction with mitochondrial RNA-binding proteins. Our findings highlight the importance of optimizing poly(A) tail length for efficient mitochondrial mRNA translation, providing a potential strategy for improving mitochondrial gene therapy. These results pave the way for further exploration of the mechanisms and clinical applications of mitochondrial mRNA delivery systems.

Brown algae play essential roles ecologically, practically, and evolutionarily because they maintain coastal areas, capture carbon dioxide, and produce valuable chemicals such as therapeutic drugs. To unlock their full potential, understanding the unique molecular biology of brown algae is imperative. Genetic engineering tools that regulate homeostasis in brown algae are essential for determining their biological mechanisms in detail. However, few methodologies have been developed to control gene expression due to the robust structural barriers of brown algae. To address this issue, we designed peptide-based, small interfering RNA (siRNA)-loaded micelles decorated with phenylboronic acid (PBA) ligands. The PBA ligands facilitated the cellular uptake of the micelles into a model brown alga, Ectocarpus siliculosus (E. Siliculosus), through chemical interaction with polysaccharides in the cell wall and biological recognition by boronic acid transporters on the plasma membrane. The micelles, featuring "kill two birds with one stone" ligands, effectively induced gene silencing related to auxin biosynthesis. As a result, the growth of E. siliculosus was temporarily inhibited without persistent genome editing. This study demonstrated the potential for exploring the characteristics of brown algae through a simple yet effective approach and presented a feasible system for delivering siRNA in brown algae.

Mitochondria are vital organelles regulating essential cellular functions. Human mitochondrial DNA (mtDNA) consists of 37 genes, 13 of which encode mitochondrial proteins, and the remaining 24 genes encode two ribosomal RNAs and 22 transfer RNAs needed for the translation of the mtDNA‐encoded 13 proteins. However, mtDNA often impairs the expression and function of these genes due to various mutations, ultimately causing mitochondrial dysfunction. To recover from this desperate condition, developing the technology to supply all mitochondrial proteins encoded by mtDNA at once is an urgent task, but there is no established strategy for this purpose. In this study, a simple yet effective mitochondrial gene delivery system is proposed comprising an artificial peptide inspired by a transmembrane mitochondrial membrane protein. The designed mitochondria‐targeting peptides presented on the carrier surface effectively guide the encapsulated plasmid to the mitochondria, facilitating mitochondrial uptake and gene expression. The developed system successfully delivers exogenous mtDNA to mtDNA‐depleted cells and leads to simultaneous multigene expression, ultimately restoring mitochondrial functions, including the mitochondrial respiration rate. The established multiple gene expression system in each mitochondrion is a game‐changing technology that can accelerate the development of mitochondrial engineering technologies as well as clinical applications for mitochondrial diseases.

Biodegradable polymers are eco-friendly materials and have attracted attention for use in a sustainable society because they are not accumulated in the environment. Although the characteristics of biodegradable polymers have been assessed well, the effects of their degradation products have not. Herein, we comprehensively evaluated the chemical toxicities of biodegradable polyester, polycaprolactone (PCL), and synthetic oligocaprolactones (OCLs) with different degrees of polymerization. While the PCL did not show any adverse effects on various organisms, high levels of shorter OCLs and the monomer (1 μg/mL for freshwater microorganisms and 1 mg/mL for marine algae and mammalian cells) damaged the tested organisms, including freshwater microorganisms, marine algae, and mammalian cells, which indicated the toxicities of the degradation products under unnaturally high concentrations. These results highlight the need for a further understanding of the effects of the degradation products resulting from biodegradable polyesters to ensure a genuinely sustainable society.

Effective delivery of the CRISPR-Cas9 components is crucial to realizing the therapeutic potential. Although many delivery approaches have been developed for this application, oral delivery has not been explored due to the degradative nature of the gastrointestinal tract. For this issue, we developed a series of novel phenylboronic acid (PBA)-functionalized chitosan-polyethylenimine (CS-PEI) polymers for oral CRISPR delivery. PBA functionalization equipped the polyplex with higher stability, smooth transport across the mucus, and efficient endosomal escape and cytosolic unpackaging in the cells. From a library of 12 PBA-functionalized CS-PEI polyplexes, we identified a formulation that showed the most effective penetration in the intestinal mucosa after oral gavage to mice. The optimized formulation performed feasible CRISPR-mediated downregulation of the target protein and reduction in the downstream cholesterol. As the first oral CRISPR carrier, this study suggests the potential of addressing the needs of both local and systemic editing in a patient-compliant manner.

Mitochondria play an essential role in cellular metabolism and generate energy in cells. To support these functions, several proteins are encoded in the mitochondrial DNA (mtDNA). The mutation of mtDNA causes mitochondrial dysfunction and ultimately results in a variety of inherited diseases. To date, gene delivery systems targeting mitochondria have been developed to ameliorate mtDNA mutations. However, applications of these strategies in mitochondrial gene therapy are still being explored and optimized. Thus, from this perspective, we herein highlight recent mitochondria-targeting strategies for gene therapy and discuss future directions for effective mitochondria-targeted gene delivery.

Messenger RNA (mRNA) delivery strategies are required to protect biologically fragile mRNA from ribonuclease (RNase) attacks to achieve efficient therapeutic protein expression. To tackle this issue, most mRNA delivery systems have used cationic components, which form electrostatically driven complexes with mRNA and shield encapsulated mRNA strands. However, cationic materials interact with anionic biomacromolecules in physiological environments, which leads to unspecific reactions and toxicities. To circumvent this issue of cation-based approaches, herein, we propose a cation-free delivery strategy by hybridization of PEGylated RNA oligonucleotides with mRNA. The PEG strands on the mRNA sterically and electrostatically shielded the mRNA, improving mRNA nuclease stability 15-fold after serum incubation compared with unhybridized mRNA. Eventually, the PEGylated mRNA induced nearly 20-fold higher efficiency of reporter protein expression than unhybridized mRNA in cultured cells. This study provides a platform to establish a safe and efficient cation-free mRNA delivery system.

For in vivo application of mRNA therapeutics, the development of mRNA nanocarriers that protect mRNA from enzymatic degradation is needed. While current nanocarrier development focuses on fine-tuning the chemical structure of its components, including lipids and polymers, herein, we propose a novel strategy to design stable mRNA nanocarriers by structuring mRNA inside the nanocarriers. Firstly, several mRNA strands were crosslinked with each other using RNA crosslinkers that hybridize to mRNA strands, to prepare mRNA nanoassemblies (NAs). Then, we mixed NAs with poly(ethylene glycol) (PEG)-polycation block copolymers to prepare core–shell-structured polyplex micelles (PMs), composed of PEG shell and mRNA-containing core. Notably, PM-loading NAs (NA/m) exhibited enhanced stability against enzymatic attack and polyion exchange reaction compared to that loading naïve mRNA (naïve/m). According to mechanistic analyses, NA/m possessed a shell with a denser PEG layer and a core with more condensed mRNA compared to naïve/m. As a result, NA/m induced more efficient protein expression after introduction to cultured cells and mouse brain, compared to naïve/m. While newly developed materials need long processes before their clinical approval, our strategy is effective in improving stability and the mRNA introduction efficiency of existing mRNA nanocarriers just by structuring mRNA without the use of additional materials.