Development of a new caged intein for multi-input conditional translation of synthetic mRNA.

mRNA medicines can be used to express therapeutic proteins, but the production of such proteins in non-target cells has a risk of adverse effects. To accurately distinguish between therapeutic target and nontarget cells, it is desirable to utilize multiple proteins expressed in each cell as indicators. To achieve such multi-input translational regulation of mRNA medicines, in this study, we engineered Rhodothermus marinus (Rma) DnaB intein to develop "caged Rma DnaB intein" that enables conditional reconstitution of full-length translational regulator protein from split fragments. By combining the caged Rma DnaB intein, the split translational regulator protein, and target protein-binding domains, we succeeded in target protein-dependent translational repression of mRNA in human cells. In addition, the caged Rma intein showed orthogonality to the previously reported Nostoc punctiforme (Npu) DnaE-based caged intein. Finally, by combining these two orthogonal caged inteins, we developed an mRNA-based logic gate that regulates translation based on the expression of multiple intracellular proteins. This study provides important information to develop safer mRNA medicines.

Intracranial Gene Delivery Mediated by Albumin-Based Nanobubbles and Low-Frequency Ultrasound.

Research in the field of high-intensity focused ultrasound (HIFU) for intracranial gene therapy has greatly progressed over the years. However, limitations of conventional HIFU still remain. That is, genes are required to cross the blood-brain barrier (BBB) in order to reach the neurological disordered lesion. In this study, we introduce a novel direct intracranial gene delivery method, bypassing the BBB using human serum albumin-based nanobubbles (NBs) injected through a less invasive intrathecal route via lumbar puncture, followed by intracranial irradiation with low-frequency ultrasound (LoFreqUS). Focusing on both plasmid DNA (pDNA) and messenger RNA (mRNA), our approach utilizes LoFreqUS for deeper tissue acoustic penetration and enhancing gene transfer efficiency. This drug delivery method could be dubbed as the "Spinal Back-Door Approach", an alternative to the "front door" BBB opening method. Experiments showed that NBs effectively responded to LoFreqUS, significantly improving gene transfer in vitro using U-87 MG cell lines. In vivo experiments in mice demonstrated significantly increased gene expression with pDNA; however, we were unable to obtain conclusive results using mRNA. This novel technique, combining albumin-based NBs and LoFreqUS offers a promising, efficient, targeted, and non-invasive solution for central nervous system gene therapy, potentially transforming the treatment landscape for neurological disorders.

Comprehensive Evaluation of Lipid Nanoparticles and Polyplex Nanomicelles for Muscle-Targeted mRNA Delivery.

The growing significance of messenger RNA (mRNA) therapeutics in diverse medical applications, such as cancer, infectious diseases, and genetic disorders, highlighted the need for efficient and safe delivery systems. Lipid nanoparticles (LNPs) have shown great promise for mRNA delivery, but challenges such as toxicity and immunogenicity still remain to be addressed. In this study, we aimed to compare the performance of polyplex nanomicelles, our original cationic polymer-based carrier, and LNPs in various aspects, including delivery efficiency, organ toxicity, muscle damage, immune reaction, and pain. Our results showed that nanomicelles (PEG-PAsp(DET)) and LNPs (SM-102) exhibited distinct characteristics, with the former demonstrating relatively sustained protein production and reduced inflammation, making them suitable for therapeutic purposes. On the other hand, LNPs displayed desirable properties for vaccines, such as rapid mRNA expression and potent immune response. Taken together, these results suggest the different potentials of nanomicelles and LNPs, supporting further optimization of mRNA delivery systems tailored for specific purposes.

Vitamin D3 Promotes Not Motor Coordination but Motor Skill Learning without Influence on Muscle Function.

Although motor coordination or motor skill learning are improved by taking vitamin D in the animal experiment, muscle function have not been estimated. Here we examined the effect of vitamin D3 administration on motor coordination and motor skill learning, muscle strength, and muscle volume in mice fed a vitamin D deficient diet. In mice fed a vitamin D deficient diet, serum calcium and 25(OH)D3 concentrations were measured. We then conducted Rotarod test, beam walking assay, micro-CT analysis, and forelimb grip strength test. Administration of vitamin D3 elongated the retention time in the Rotarod test in a time dependent manner. In contrast, the time to reach a beam goal box in beam walking assay was not changed in mice administered with vitamin D3, compared to the control. Oral administration of vitamin D3 did not affect muscle strength nor muscle volume. Oral administration of vitamin D3 promotes not motor coordination but motor skill learning and does not affect muscle function.

Administration of mRNA-Nanomedicine-Augmented Calvarial Defect Healing via Endochondral Ossification.

Large-area craniofacial defects remain a challenge for orthopaedists, hastening the need to develop a facile and safe tissue engineering strategy; osteoconductive material and a combination of optimal growth factors and microenvironment should be considered. Faced with the unmet need, we propose that abundant cytokines and chemokines can be secreted from the bone defect, provoking the infiltration of endogenous stem cells to assist bone regeneration. We can provide a potent mRNA medicine cocktail to promptly initiate the formation of bone templates, osteogenesis, and subsequent bone matrix deposition via endochondral ossification, which may retard rapid fibroblast infiltration and prevent the formation of atrophic non-union. We explored the mutual interaction of BMP2 and TGFß3 mRNA, both potent chondrogenic factors, on inducing endochondral ossification; examined the influence of in vitro the transcribed polyA tail length on mRNA stability; prepared mRNA nanomedicine using a PEGylated polyaspartamide block copolymer loaded in a gelatin sponge and grafted in a critical-sized calvarial defect; and evaluated bone regeneration using histological and µCT examination. The BMP2 and TGFß3 composite mRNA nanomedicine resulted in over 10-fold new bone volume (BV) regeneration in 8 weeks than the BMP2 mRNA nanomedicine administration alone, demonstrating that the TGFß3 mRNA nanomedicine synergistically enhances the bone's formation capability, which is induced by BMP2 mRNA nanomedicine. Our data demonstrated that mRNA-medicine-mediated endochondral ossification provides an alternative cell-free tissue engineering methodology for guiding craniofacial defect healing.

Enhancement of bone regeneration by coadministration of angiogenic and osteogenic factors using messenger RNA.

BACKGROUND: Bone defects remain a challenge today. In addition to osteogenic activation, the crucial role of angiogenesis has also gained attention. In particular, vascular endothelial growth factor (VEGF) is likely to play a significant role in bone regeneration, not only to restore blood supply but also to be directly involved in the osteogenic differentiation of mesenchymal stem cells. In this study, to produce additive angiogenic-osteogenic effects in the process of bone regeneration, VEGF and Runt-related transcription factor 2 (Runx2), an essential transcription factor for osteogenic differentiation, were coadministered with messenger RNAs (mRNAs) to bone defects in the rat mandible. METHODS: The mRNAs encoding VEGF or Runx2 were prepared via in vitro transcription (IVT). Osteogenic differentiation after mRNA transfection was evaluated using primary osteoblast-like cells, followed by an evaluation of the gene expression levels of osteogenic markers. The mRNAs were then administered to a bone defect prepared in the rat mandible using our original cationic polymer-based carrier, the polyplex nanomicelle. The bone regeneration was evaluated by micro-computerized tomography (µCT) imaging, and histologic analyses. RESULTS: Osteogenic markers such as osteocalcin (Ocn) and osteopontin (Opn) were significantly upregulated after mRNA transfection. VEGF mRNA was revealed to have a distinct osteoblastic function similar to that of Runx2 mRNA, and the combined use of the two mRNAs resulted in further upregulation of the markers. After in vivo administration into the bone defect, the two mRNAs induced significant enhancement of bone regeneration with increased bone mineralization. Histological analyses using antibodies against the Cluster of Differentiation 31 protein (CD31), alkaline phosphatase (ALP), or OCN revealed that the mRNAs induced the upregulation of osteogenic markers in the defect, together with increased vessel formation, leading to rapid bone formation. CONCLUSIONS: These results demonstrate the feasibility of using mRNA medicines to introduce various therapeutic factors, including transcription factors, into target sites. This study provides valuable information for the development of mRNA therapeutics for tissue engineering.

Efficient mRNA Delivery with Lyophilized Human Serum Albumin-Based Nanobubbles.

In this study, we developed an efficient mRNA delivery vehicle by optimizing a lyophilization method for preserving human serum albumin-based nanobubbles (HSA-NBs), bypassing the need for artificial stabilizers. The morphology of the lyophilized material was verified using scanning electron microscopy, and the concentration, size, and mass of regenerated HSA-NBs were verified using flow cytometry, nanoparticle tracking analysis, and resonance mass measurements, and compared to those before lyophilization. The study also evaluated the response of HSA-NBs to 1 MHz ultrasound irradiation and their ultrasound (US) contrast effect. The functionality of the regenerated HSA-NBs was confirmed by an increased expression of intracellularly transferred Gluc mRNA, with increasing intensity of US irradiation. The results indicated that HSA-NBs retained their structural and functional integrity markedly, post-lyophilization. These findings support the potential of lyophilized HSA-NBs, as efficient imaging, and drug delivery systems for various medical applications.

Development of Synthetic mRNAs Encoding Split Cytotoxic Proteins for Selective Cell Elimination Based on Specific Protein Detection.

For the selective elimination of deleterious cells (e.g., cancer cells and virus-infected cells), the use of a cytotoxic gene is a promising approach. DNA-based systems have achieved selective cell elimination but risk insertional mutagenesis. Here, we developed a synthetic mRNA-based system to selectively eliminate cells expressing a specific target protein. The synthetic mRNAs used in the system are designed to express an engineered protein pair that are based on a cytotoxic protein, Barnase. Each engineered protein is composed of an N- or C-terminal fragment of Barnase, a target protein binding domain, and an intein that aids in reconstituting full-length Barnase from the two fragments. When the mRNAs are transfected to cells expressing the target protein, both N- and C-terminal Barnase fragments bind to the target protein, causing the intein to excise itself and reconstitute cytotoxic full-length Barnase. In contrast, when the target protein is not present, the reconstitution of full-length Barnase is not induced. Four candidate constructs containing split Barnase were evaluated for the ability to selectively eliminate target protein-expressing cells. One of the candidate sets demonstrated highly selective cell death. This system will be a useful therapeutic tool to selectively eliminate deleterious cells.

Unbiased comparison and modularization identify time-related transcriptomic reprogramming in exercised rat cartilage: Integrated data mining and experimental validation.

Exercise is indispensable for maintaining cartilage integrity in healthy joints and remains a recommendation for knee osteoarthritis. Although the effects of exercise on cartilage have been implied, the detailed mechanisms, such as the effect of exercise time which is important for exercise prescription, remain elusive. In this study, bioinformatic analyses, including unbiased comparisons and modularization, were performed on the transcriptomic data of rat cartilage to identify the time-related genes and signaling pathways. We found that exercise had a notable effect on cartilage transcriptome. Exercise prominently suppressed the genes related to cell division, hypertrophy, catabolism, inflammation, and immune response. The downregulated genes were more prominent and stable over time than the upregulated genes. Although exercise time did not prominently contribute to the effects of exercise, it was a factor related to a batch of cellular functions and signaling pathways, such as extracellular matrix (ECM) homeostasis and cellular response to growth factors and stress. Two clusters of genes, including early and late response genes, were identified according to the expression pattern over time. ECM organization, BMP signaling, and PI3K-Akt signaling were early responsive in the exercise duration. Moreover, time-related signaling pathways, such as inositol phosphate metabolism, nicotinate/nicotinamide metabolism, cell cycle, and Fc epsilon RI signaling pathway, were identified by unbiased mapping and polarization of the highly time-correlated genes. Immunohistochemistry staining showed that Egfr was a late response gene that increased on day 15 of exercise. This study elucidated time-related transcriptomic reprogramming induced by exercise in cartilage, advancing the understanding of cartilage homeostasis.

Anti-Inflammatory Therapy for Temporomandibular Joint Osteoarthritis Using mRNA Medicine Encoding Interleukin-1 Receptor Antagonist.

Messenger RNA (mRNA) is an emerging drug modality for protein replacement therapy. As mRNA efficiently provides protein expression in post-mitotic cells without the risk of insertional mutagenesis, direct delivery of mRNA can be applied, not only as an alternative to gene therapy, but also for various common diseases such as osteoarthritis (OA). In this study, using an mRNA-encoding interleukin-1 receptor antagonist (IL-1Ra), we attempted anti-inflammatory therapy in a rat model of the temporomandibular joint (TMJ) OA, which causes long-lasting joint pain with chronic inflammation. For the intra-articular injection of mRNA, a polyplex nanomicelle, our original polymer-based carrier, was used to offer the advantage of excellent tissue penetration with few immunogenic responses. While the protein expression was transient, a single administration of IL-1Ra mRNA provided sustained pain relief and an inhibitory effect on OA progression for 4 weeks. The mRNA-loaded nanomicelles provided the encoded protein diffusely in the disc and articular cartilage without upregulation of the expression levels of the pro-inflammatory cytokines IL-6 and tumor necrosis factor-α (TNF-α). This proof-of-concept study demonstrates how anti-inflammatory proteins delivered by mRNA delivery using a polyplex nanomicelle could act to alleviate OA, stimulating the development of mRNA therapeutics.

Brain Dp140 alters glutamatergic transmission and social behaviour in the mdx52 mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a muscle disorder caused by DMD mutations and is characterized by neurobehavioural comorbidities due to dystrophin deficiency in the brain. The lack of Dp140, a dystrophin short isoform, is clinically associated with intellectual disability and autism spectrum disorders (ASDs), but its postnatal functional role is not well understood. To investigate synaptic function in the presence or absence of brain Dp140, we utilized two DMD mouse models, mdx23 and mdx52 mice, in which Dp140 is preserved or lacking, respectively. ASD-like behaviours were observed in pups and 8-week-old mdx52 mice lacking Dp140. Paired-pulse ratio of excitatory postsynaptic currents, glutamatergic vesicle number in basolateral amygdala neurons, and glutamatergic transmission in medial prefrontal cortex-basolateral amygdala projections were significantly reduced in mdx52 mice compared to those in wild-type and mdx23 mice. ASD-like behaviour and electrophysiological findings in mdx52 mice were ameliorated by restoration of Dp140 following intra-cerebroventricular injection of antisense oligonucleotide drug-induced exon 53 skipping or intra-basolateral amygdala administration of Dp140 mRNA-based drug. Our results implicate Dp140 in ASD-like behaviour via altered glutamatergic transmission in the basolateral amygdala of mdx52 mice.

Influence of Nanobubble Size Distribution on Ultrasound-Mediated Plasmid DNA and Messenger RNA Gene Delivery.

The use of nanobubbles (NBs) for ultrasound-mediated gene therapy has recently attracted much attention. However, few studies have evaluated the effect of different NB size distribution to the efficiency of gene delivery into cells. In this study, various size of albumin stabilized sub-micron bubbles were examined in an in vitro ultrasound (1 MHz) irradiation setup in the aim to compare and optimize gene transfer efficiency. Results with pDNA showed that gene transfer efficiency in the presence of NB size of 254.7 ± 3.8 nm was 2.5 fold greater than those with 187.3 ± 4.8 nm. Similarly, carrier-free mRNA transfer efficiency increased in the same conditions. It is suggested that NB size greater than 200 nm contributed more to the delivery of genes into the cytoplasm with ultrasound. Although further experiments are needed to understand the underlying mechanism for this phenomenon, the present results offer valuable information in optimizing of NB for future ultrasound-mediate gene therapy.

Synthetic mRNA for ex vivo therapeutic applications.

Synthetic mRNA is attracting much attention as a new drug modality. Now, there is great interest in the next applications of mRNA medicines, especially for therapeutic purposes of various diseases. Not only in vivo applicable mRNA medicines, there are many researches using ex vivo or in vitro mRNA transfection, some of which are already used in the preclinical or clinical settings. In this short article, the ex vivo mRNA applications are reviewed, in the hope of providing insight to develop future mRNA medicines.

Raman spectroscopic insight into osteoarthritic cartilage regeneration by mRNA therapeutics encoding cartilage-anabolic transcription factor Runx1.

While joint arthroplasty remains nowadays the most popular option available to repair chronically degenerated osteoarthritic joints, possibilities are recently emerging for regeneration of damaged cartilage rather than its replacement with artificial biomaterials. This latter strategy could allow avoiding the quite intrusive surgical procedures associated with total joint replacement. Building upon this notion, we first apply Raman spectroscopy to characterize diseased cartilage in a mice model of instability-induced knee osteoarthritis (OA) upon medial collateral ligament (MCL) and medial meniscus (MM) transections. Then, we examine the same OA model after cartilage regeneration by means of messenger RNA (mRNA) delivery of a cartilage-anabolic runt-related transcription factor 1 (RUNX1). Raman spectroscopy is shown to substantiate at the molecular scale the therapeutic effect of the Runx1 mRNA cartilage regeneration approach. This study demonstrates how the Raman spectroscopic method could support and accelerate the development of new therapies for cartilage diseases.

Versatile Design of Intracellular Protein-Responsive Translational Regulation System for Synthetic mRNA.

Synthetic mRNA (mRNA) enables transgene expression without the necessity of nuclear import and the risk of insertional mutagenesis, which makes it an attractive tool for medical applications such as vaccination and protein replacement therapy. For further improvement of mRNA therapeutics, cell-selective translation is desirable, because transgene expression in nontarget cells sometimes causes adverse effects. In this study, we developed an intracellular protein-responsive translational regulation system based on Caliciviral VPg-based translational activator (CaVT) combined with inteins and target protein-binding nanobodies. This system enabled both translational activation and repression in a target protein-dependent manner. Importantly, the target protein can be altered by simply exchanging the nanobodies. The versatile design for target protein-responsive translational regulation holds promise for producing mRNA therapeutics with high safety.

Efficient Messenger RNA Delivery to the Kidney Using Renal Pelvis Injection in Mice.

Renal dysfunction is often associated with the inflammatory cascade, leading to non-reversible nephrofibrosis. Gene therapy has the ability to treat the pathology. However, the difficulty in introducing genes into the kidney, via either viral vectors or plasmid DNA (pDNA), has hampered its extensive clinical use. Messenger RNA (mRNA) therapeutics has recently attracted attention as alternative gene therapies. mRNA allows protein production into post-mitotic cells without the need for transport to the nuclei in the target cells. However, few studies have reported the delivery of mRNA to the kidney. In this study, we attempted to deliver mRNA to the kidney based on the principle of pressure stimulation, by administering mRNA-loaded polyplex nanomicelles via a renal pelvis injection, directly into the kidney. Compared with the administration of naked plasmid DNA (pDNA) and naked mRNA, the mRNA-loaded nanomicelles diffusely induced protein expression in a greater number of cells at the tubular epithelium for some days. The plasma creatinine (Cre) and blood urea nitrogen (BUN) levels after the administration remained similar to those of the sham-operated controls, without marked changes in histological sections. The safety and efficacy of mRNA-loaded nanomicelles would make distinct contributions to the development of mRNA therapeutics for the kidney.

Treatment of ischemic neuronal death by introducing brain-derived neurotrophic factor mRNA using polyplex nanomicelle.

Ischemic neuronal death causes serious lifelong neurological deficits; however, there is no proven effective treatment that can prevent neuronal death after the ischemia. We investigated the feasibility of mRNA therapeutics for preventing the neuronal death in a rat model of transient global ischemia (TGI). By intraventricular administration of mRNA encoding brain-derived neurotrophic factor (BDNF) using a polymer-based carrier, polyplex nanomicelle, the mRNA significantly increased the survival rate of hippocampal neurons after TGI, with a rapid rise of BDNF in the hippocampus. Interestingly, mRNA administration on Day 2 after TGI provided significantly better survival rate than the administration immediately after TGI. Eventually, dosing twice on Day 2 and 5 exerted long-term therapeutic effects, which were confirmed by a Y-maze behavioral test demonstrating improved spatial memory compared with untreated rats on Day 20. Immunohistochemical analysis showed that astrocytes were chief targets of the BDNF mRNA-loaded nanomicelles, suggesting that the augmented BDNF secretion from astrocytes creates a supportive microenvironment for the neurons to tolerate changes caused by ischemic stresses, and terminate the process of progressive neuronal death after the ischemic attack. Overall, the unique mechanism of action of mRNA therapeutics provide a promising approach for preventing ischemic neuronal death.

Regulation of Chondrocyte Differentiation by Changing Intercellular Distances Using Type II Collagen Microfibers.

Osteoarthritis is a common degenerative disease that mainly occurs in older age groups, and the search for an effective cure remains a major global challenge. The technology of constructing 3D in vitro cartilage tissue with zonal differentiated structures for use as alternative implants for treating osteoarthritis has attracted researchers' attention. For this challenge, it is important for understanding the relationship between chondrocyte differentiation and the amount of extracellular matrix by modulating intercellular distance. This study investigates the interplay between chondrocyte differentiation and intercellular distance. Type II collagen microfibers (CMF II) were used as a distance regulator by varying their amounts. The results indicated that the secretion of cartilage-specific glycosaminoglycan after 2 weeks of differentiation from the chondrogenic cells, ATDC5, was decreased with an increased intercellular distance. Also, the shortest intercellular distance, being ATDC5 cells without CMF II, presented an upregulated gene expression profile of cartilage markers. The groups with CMF II-mediated intracellular distances, however, did not show the upregulation. The elastic modulus of the 3D samples increased depending on the amount of CMF II, relating to the differentiation preventing property of the CMF II. These findings suggest the promising potential of this approach for the modulation of chondrocyte differentiation.

mRNA as a Tool for Gene Transfection in 3D Cell Culture for Future Regenerative Therapy.

A combination of three-dimensional (3D) cell culturing and non-viral gene transfection is promising in improving outcomes of cell transplantation therapy. Herein, gene transfection profiles in 3D cell culture were compared between plasmid DNA (pDNA) and messenger RNA (mRNA) introduction, using mesenchymal stem cell (MSC) 3D spheroids. Green fluorescence protein (GFP) mRNA induced GFP protein expression in 77% of the cells in the spheroids, whereas only 34% of the cells became GFP positive following pDNA introduction. In mechanistic analyses, most of the cells in MSC spheroids were non-dividing, and pDNA failed to induce GFP expression in most of the non-dividing cells. In contrast, both dividing and non-dividing cells became GFP-positive after mRNA introduction, which led to a high overall percentage of GFP-positive cells in the spheroids. Consequently, mRNA encoding an osteogenic factor, runt-related transcription factor 2 (Runx2), allowed in vitro osteogenic differentiation of MSCs in spheroids more efficiently compared to Runx2 pDNA. Conclusively, mRNA exhibits high potential in gene transfection in 3D cell culture, in which the cell division rate is lower than that in monolayer culture, and the combination of mRNA introduction and 3D cell culture is a promising approach to improve outcomes of cell transplantation in future regenerative therapy.