Biology subjects

Kawai-Harada, Y.

Publications and source records attributed to Kawai-Harada, Y..

6 recordsLinked to original sources

bioRxiv2025

A scalable filtration-based method for isolating exomeres and other nanoscale extracellular particles

Extracellular particles, including extracellular vesicles (EVs) and non-vesicular extracellular particles (NVEPs), enable intercellular communication by transferring regulatory miRNAs and other biomolecules. While EVs have been studied for drug delivery, NVEPs remain relatively unexplored. Exomeres, a recently discovered class of NVEPs enriched in RNAi proteins, preferentially carry miRNAs and deliver them to cells more effectively than EVs, underscoring their potential as vehicles for therapeutic RNAs. One current limitation to studying and applying exomeres for therapeutic RNA delivery is the shortage of scalable, cost-effective, and rapid isolation methods. Here, we investigated whether tangential flow filtration (TFF), a common bioseparation approach that separates species by size, would effectively isolate exomeres from conditioned media with comparable purity and identity to those isolated by differential ultracentrifugation. TFF successfully isolated exomeres that were enriched in RNAi components including AGO2, HSP90AB1, and a unique set of miRNAs not abundant in EVs. Remarkably, exomere miRNAs were resistant to nuclease degradation even after treatment with protease and surfactant, suggesting that exomeres are highly stable, non-vesicular complexes with potentially extended circulating half-lives. Together, our results establish TFF as an efficient method for isolating exomeres, and further demonstrate that TFF could be applied in a bioprocess for exomere-based RNA therapeutics production. This study is also the first to demonstrate that exomere miRNAs are highly resistant to nuclease degradation, suggesting that exomeres could complement and potentially outperform current clinical standards for RNA delivery.

bioengineering↗

bioRxiv2025

Generation of Cellular Biofactories for Scalable Production of Surface-Engineered Extracellular Vesicles via CRISPR Genome Editing

Extracellular vesicles (EVs) are versatile biological nanoparticles with applications in therapeutics, diagnostics, and biotechnology. Current production methods using transient transfection or chemical conjugation suffer from high variability, limited scalability, and heterogeneous EV populations. Here, we developed CRISPR-Cas9 engineered HEK293T cell lines with stable integration of mCherry-C1C2 fusion proteins at the AAVS1 locus for continuous production of surface-modified EVs. The engineered cell lines demonstrated significantly higher surface display efficiency compared to transient transfection, with reduced batch-to-batch variability. EVs maintained native characteristics including size distribution (120-130 nm) and marker expression while showing efficient cellular uptake. The platform maintained consistent production of uniformly modified EVs with stable transgene expression over at least 25 passages (~3 months), eliminating the need for repeated transfections and reducing batch-to-batch variability inherent to transient expression systems.

bioengineering↗

bioRxiv2025

VesicleVoyager: In vivo selection of surface displayed proteins that direct extracellular vesicles to tissue-specific targets

The development of technologies for screening proteins that bind to specific tissues in vivo and facilitate delivery of large cargos remains challenging, with most approaches limited to cell culture systems that often yield clinically irrelevant hits. To overcome this limitation, we developed a novel molecular screening platform using an extracellular vesicle (EV) display library. EVs are natural molecular carriers capable of delivering diverse cargos, which can be engineered to enhance specificity and targeting through surface modifications. We constructed an EV-display library presenting monobody repertoires on EV surfaces, with genetic cargo inside the EVs corresponding to the displayed proteins. These libraries were screened for tissue specific delivery through serial passage in mice via sequential intravenous administration in and recovery of tissue-selected EVs and amplification of their encapsulated monobody genes at each passage. Our results demonstrated successful selection of tissue-specific targeting proteins, as revealed by fluorescence and bioluminescence imaging followed by DNA sequencing. To understand the stochastic relationship between displayed proteins and packaged genes, we developed a Markov chain model that quantified selection dynamics and predicted enrichment patterns despite the imperfect correlation between phenotype and genotype. This EV-based monobody screening approach, combined with mathematical modeling, is a significant advancement in targeted drug delivery by leveraging the natural capabilities of EVs with the selection of targeting proteins in a physiologically relevant environment.

bioengineering↗

bioRxiv2024

RecombiCraft Library construction: A novel method for DNA Library cloning and expansion using non-enzymatic single-step DNA recombination and liquid culture

In this study, we introduce RecombiCraft, an innovative, rapid, and cost-efficient method for constructing DNA libraries in E. coli. This method uses seamless ligation cloning extract (SLiCE) coupled with liquid culture amplification to effectively minimize sequence biases. The technique capitalizes on the natural homologous recombination capabilities of E. coli cell lysates, eliminating the need for multiple purified enzymes and reducing costs. We first synthesized the library backbone and inserts via PCR, employing high-fidelity polymerase to minimize sequence bias. The SLiCE technique was then used to assemble the DNA fragments introduced into E. coli through electroporation. To ensure the integrity of the library, we optimized culture times based on next-generation sequencing analysis which confirmed the minimal sequence bias. The RecombiCraft method demonstrates that this approach is economical and maintains the librarys uniformity. It is a promising tool for genetic research and biotechnological applications with a significantly shorter library generation period.

molecular biology↗

bioRxiv2024

Scalable Isolation of Surface-Engineered Extracellular Vesicles and Separation of Free Proteins via Tangential Flow Filtration and Size Exclusion Chromatography (TFF-SEC)

BackgroundExtracellular vesicles (EVs) represent small lipid bilayer structures pivotal in mediating intercellular communication via biomolecular transfer. Their inherent characteristics, including packaging, non-immunogenicity, and biofluid stability, position EVs as promising drug delivery vectors. However, developing clinical quality EVs requires multifaceted technological advancement. MethodsIn this study, a method is introduced for engineering extracellular vesicles (eEVs) from cultured cells and their subsequent isolation using lab-scale tangential flow filtration (TFF). This is the first study to evaluate DNA loading efficacy into EVs isolated by TFF, marking a significant milestone in the field of targeted drug delivery. Initially, cells are transfected with EV-display constructs to facilitate the secretion of eEVs bearing the desired coding molecules. Following brief centrifugation, the cell culture media undergoes filtration using hollow fiber filters. TFF, by applying a constant flow, effectively segregates molecules based on designated molecular weight cut-off (MWCO), enriching particles between 50 nm and 650 nm. ResultsCompared to conventional methods like ultracentrifugation, TFF demonstrates higher efficiency in removing undesired molecules/aggregates while exerting less stress on EVs. Characterization of eEVs through various assays confirms TFFs superiority in isolating pure EV populations. Additionally, the necessity of size-exclusion chromatography (SEC) after tangential flow filtration (TFF) becomes evident for effectively removing unbound protein contaminants. ConclusionIn conclusion, TFF-SEC emerges as a scalable and superior approach for eEV isolation, promising significant advancements in clinical applications.

bioengineering↗

bioRxiv2022

Evaluation of EV Storage Buffer for Efficient Preservation of Engineered Extracellular Vesicles

Extracellular vesicles (EVs), detectable in all bodily fluids, mediate intercellular communication by transporting molecules between cells. The capacity of EVs to transport molecules between distant organs has drawn interest for clinical applications in diagnostics and therapeutics. Although EVs hold potential for nucleic acid-based and other molecular therapeutics, the lack of standardized technologies, including isolation, characterization, and storage, leaves many challenges for clinical applications, potentially resulting in misinterpretation of crucial findings. Previously, several groups demonstrated the problems of commonly used storage methods that distort EV integrity. This work aims to evaluate the process to optimize the storage conditions of EVs and then characterize them according to the experimental conditions and the models used previously. Our study reports a highly efficient EV storage condition, focusing on EVs capacity to protect their molecular cargo from biological, chemical, and mechanical damage. Compared with commonly used EV storage conditions, our EV storage buffer leads to less size and particle number variation at both 4{degrees}C and -80 {degrees}C, enhancing the ability to protect EVs while maintaining targeting functionality.

bioengineering↗