【公開日：2025.06.10】【最終更新日：2025.05.21】
課題データ / Project Data
課題番号 / Project Issue Number
24UT0220
利用課題名 / Title
Drug delivery systems
利用した実施機関 / Support Institute
東京大学 / Tokyo Univ.
機関外・機関内の利用 / External or Internal Use
外部利用/External Use
技術領域 / Technology Area
【横断技術領域 / Cross-Technology Area】（主 / Main）計測・分析/Advanced Characterization（副 / Sub）-
【重要技術領域 / Important Technology Area】（主 / Main）次世代バイオマテリアル/Next-generation biomaterials（副 / Sub）マルチマテリアル化技術・次世代高分子マテリアル/Multi-material technologies / Next-generation high-molecular materials
キーワード / Keywords
電子顕微鏡/ Electronic microscope,DDSマテリアル/ DDS material,Adeno-associated virus, Self-assembly, Tannic acid, Boronic acid, Block copolymer
利用者と利用形態 / User and Support Type
利用者名（課題申請者）/ User Name (Project Applicant)
Dirisala Anjaneyulu
所属名 / Affiliation
公益財団法人川崎市産業振興財団 ナノ医療イノベーションセンター（iCONM）
共同利用者氏名 / Names of Collaborators in Other Institutes Than Hub and Spoke Institutes
Yuto Honda,Shuhei Nagao,Hiroaki Kinoh,Nobuhiro Nishiyama
ARIM実施機関支援担当者 / Names of Collaborators in The Hub and Spoke Institutes
Institute of Science Tokyo,Innovation Center of Nanomedicine (iCONM)
利用形態 / Support Type
（主 / Main）技術補助/Technical Assistance（副 / Sub）,機器利用/Equipment Utilization
利用した主な設備 / Equipment Used in This Project
報告書データ / Report
概要（目的・用途・実施内容）/ Abstract (Aim, Use Applications and Contents)
Adeno-associated viruses (AAVs) have emerged as powerful vectors in gene therapy for treating severe genetic disorders. However, their systemic administration faces two critical challenges: neutralization by pre-existing antibodies (NAbs) and dose-dependent hepatotoxicity, which hinder their therapeutic efficacy. To overcome these barriers, we introduce a novel sequential assembly strategy utilizing tannic acid (TA) and phenylboronic acid-conjugated polymers to create AAV-loaded ternary complexes with a core-shell architecture, averaging 60 nm in diameter in aqueous solution.In this design, TA effectively coats AAVs and forms boronate ester linkages with boronic acid-functionalized polymers, allowing the encapsulation of AAV serotype 9 (AAV9, ~25 nm) within a protective polymeric shell. This shielding mechanism enables AAV9 to evade immune inactivation while reducing hepatic accumulation, thereby mitigating hepatotoxicity. Upon intravenous administration, the ternary complexes successfully bypass NAbs and enhance gene delivery efficiency by facilitating intracellular AAV9 release. Importantly, they preserve AAV9’s intrinsic ability to cross the blood-brain barrier (BBB), achieving a remarkable 20-fold increase in brain-to-liver transduction selectivity compared to unmodified AAV9.Furthermore, integrating this supramolecular approach with a microbubble-focused ultrasound (MB-FUS) system for noninvasive BBB disruption dramatically amplifies brain-targeted gene transduction, enhancing efficiency by over sixfold while improving brain/liver selectivity. This synergistic combination of nanotechnology and medical device innovation represents a transformative advancement in AAV-based gene therapy, offering a highly effective strategy for precise and safe genetic interventions.
実験 / Experimental
Three microliters of AAV9 (5.0 × 10¹² vg/mL) in 1 mM HEPES (pH 7.4) were carefully deposited onto a 200-mesh copper grid coated with a carbon film. Excess solution was blotted for 3 seconds using filter paper in an automatic plunge freezer (EM GP, Leica Microsystems, Wetzlar, Germany) under controlled conditions (95% humidity, 24 °C) to create a uniform thin film of AAV9 solution. The grid was then rapidly plunged into liquid ethane at −175 °C for shock-freezing. Subsequently, the vitrified sample was transferred to a JEM-2100F cryo-TEM (JEOL, Tokyo) using a Gatan 914 side-entry cryo-transfer holder (Gatan Inc., Pleasanton, CA) and imaged at an accelerated voltage of 200 kV.
結果と考察 / Results and Discussion
In this study, we engineered a systemically applicable AAV delivery platform through sequential self-assembly with tannic acid (TA) and phenylboronic acid-conjugated polymers. Cryo-electron microscopy (cryo-EM) revealed that both AAV9 and AAV9/TA/Polymer exhibited a spherical and well-dispersed morphology, whereas AAV9/TA displayed a heterogeneous and irregular shape, consistent with variations in polydispersity index values observed using Dynamic Light Scattering (Figure 1). Notably, the AAV9/TA/Polymer complex demonstrated successful encapsulation of a single AAV9 particle within its structure, highlighting the stability and uniformity of the formulation. In this study, we developed a systemically applicable AAV delivery platform by leveraging sequential self-assembly with tannic acid (TA) and phenylboronic acid-conjugated polymers. This nanocomplex effectively shielded AAV9 from neutralizing antibodies, significantly enhancing its stability and therapeutic potential. Moreover, the system improved gene transduction efficiency in the brain and targeted sites while minimizing off-target expression in organs such as the liver and kidneys, thereby achieving highly selective brain transduction and mitigating hepatotoxicity. Additionally, the integration of ultrasound irradiation further amplified gene delivery efficiency to the brain. This advanced AAV delivery strategy, in combination with a medical device, represents a transformative approach for systemic gene therapy applications.
図・表・数式 / Figures, Tables and Equations
FIgure 1. Cryo-EM images of AAV9, AAV9 coated with tannic acid (AAV9/TA), and AAV9 coated with both tannic acid and PEG-P[Lys/Lys(FPBA)] (AAV9/TA/polymer)
その他・特記事項（参考文献・謝辞等） / Remarks(References and Acknowledgements)
We thank Dr. Ayumi Kimura of ARIM at the University of Tokyo for observing cryo-EM images.
成果発表・成果利用 / Publication and Patents
論文・プロシーディング（DOIのあるもの） / DOI (Publication and Proceedings)
-
Yuto Honda, Adeno-Associated Virus Self-Assembled with Tannic Acid and Phenylboronic Acid Polymers to Evade Neutralizing Antibodies and Reduce Adverse Events, ACS Nano, 19, 7690-7706(2025).
DOI: 10.1021/acsnano.4c11085
口頭発表、ポスター発表および、その他の論文 / Oral Presentations etc.
特許 / Patents
特許出願件数 / Number of Patent Applications：1件
特許登録件数 / Number of Registered Patents：1件