Presentation Information
[P01-048]Engineering a HER2-Targeted Artificial Protein Nucleocapsid for Selective RNA Delivery
○Tomoaki Kanazawa1, Tongil Ko1, Kilian Colas1 (1. University of Tokyo (Japan))
Keywords:
Protein Engineering,RaPID Selection,Lasso-grafting,Self-assembly of capsids,RNA Delivery
[Purpose]
RNA delivery is essential for nucleic acid therapeutics, but efficient and cell-selective delivery remains a major challenge. Although viral vectors and lipid nanoparticles are widely used, protein-based nonviral carriers offer advantages such as biocompatibility, modularity, and ease of engineering. In this study, we aimed to develop HER2-targeted artificial nucleocapsids for selective RNA delivery by incorporating a HER2-binding peptide into an evolvable nonviral capsid platform. HER2 was chosen as the target receptor because it is a clinically important cell-surface marker that is highly expressed in several cancers, making it an attractive target for selective RNA delivery. It also provides a suitable model for testing whether receptor-binding peptides displayed on artificial nucleocapsids can confer target-specific binding and potentially enhance cellular uptake.
[Method]
The delivery scaffold used in this study was NC4, an artificial nucleocapsid derived from Aquifex aeolicus lumazine synthase. NC4 packages its own BoxB-tagged mRNA in E. coli through the interaction between a lumenally displayed λN-derived RNA-binding motif and the BoxB RNA stem-loop, thereby linking genotype and phenotype within the same particle. To introduce HER2-targeting ability, we first performed RaPID selection and identified a HER2-binding peptide, termed aKT0, which showed a binding affinity of 9.95 nM toward HER2. This peptide was then incorporated into NC4-based constructs using two strategies. In one strategy, a DogTag was genetically introduced into a surface loop of NC4 and the partner protein DogCatcher was co-expressed, enabling exterior peptide display through the DogTag/DogCatcher system. In the other strategy, aKT0 was directly lasso-grafted into an exposed loop of NC4 without using DogTag/DogCatcher.
[Results]
RaPID selection identified aKT0 as the top-hit peptide against HER2, with a binding affinity of 9.95 nM. Using this peptide sequence, two HER2-targeted NC4 variants were constructed through a DogTag/DogCatcher-based display system and direct lasso-grafting, respectively. Both capsid variants were confirmed to exhibit high binding activity toward HER2. Further characterization of these constructs, including TEM analysis and RNA packaging efficiency, is currently ongoing.
[Consideration]
The direct lasso-grafting strategy was considered particularly promising because the DogTag/DogCatcher system introduces a relatively long coding sequence, which may reduce mRNA packaging or translation efficiency. In contrast, direct grafting shortens the encoded sequence and may improve expression, structural stability, and suitability for directed evolution. Comparing these two display formats is expected to clarify how surface engineering strategies influence the performance of receptor-targeted artificial nucleocapsids.
[Conclusion]
This study provides a foundation for the development of HER2-targeted artificial nucleocapsids as nonviral RNA delivery vehicles. By comparing two surface-display strategies, it offers insight into how exterior engineering influences the properties of receptor-targeted nucleocapsids. In the long term, if customizable RNA loading can be achieved, this platform may support the development of versatile and evolvable protein-based carriers for targeted delivery of diverse therapeutic RNAs.
RNA delivery is essential for nucleic acid therapeutics, but efficient and cell-selective delivery remains a major challenge. Although viral vectors and lipid nanoparticles are widely used, protein-based nonviral carriers offer advantages such as biocompatibility, modularity, and ease of engineering. In this study, we aimed to develop HER2-targeted artificial nucleocapsids for selective RNA delivery by incorporating a HER2-binding peptide into an evolvable nonviral capsid platform. HER2 was chosen as the target receptor because it is a clinically important cell-surface marker that is highly expressed in several cancers, making it an attractive target for selective RNA delivery. It also provides a suitable model for testing whether receptor-binding peptides displayed on artificial nucleocapsids can confer target-specific binding and potentially enhance cellular uptake.
[Method]
The delivery scaffold used in this study was NC4, an artificial nucleocapsid derived from Aquifex aeolicus lumazine synthase. NC4 packages its own BoxB-tagged mRNA in E. coli through the interaction between a lumenally displayed λN-derived RNA-binding motif and the BoxB RNA stem-loop, thereby linking genotype and phenotype within the same particle. To introduce HER2-targeting ability, we first performed RaPID selection and identified a HER2-binding peptide, termed aKT0, which showed a binding affinity of 9.95 nM toward HER2. This peptide was then incorporated into NC4-based constructs using two strategies. In one strategy, a DogTag was genetically introduced into a surface loop of NC4 and the partner protein DogCatcher was co-expressed, enabling exterior peptide display through the DogTag/DogCatcher system. In the other strategy, aKT0 was directly lasso-grafted into an exposed loop of NC4 without using DogTag/DogCatcher.
[Results]
RaPID selection identified aKT0 as the top-hit peptide against HER2, with a binding affinity of 9.95 nM. Using this peptide sequence, two HER2-targeted NC4 variants were constructed through a DogTag/DogCatcher-based display system and direct lasso-grafting, respectively. Both capsid variants were confirmed to exhibit high binding activity toward HER2. Further characterization of these constructs, including TEM analysis and RNA packaging efficiency, is currently ongoing.
[Consideration]
The direct lasso-grafting strategy was considered particularly promising because the DogTag/DogCatcher system introduces a relatively long coding sequence, which may reduce mRNA packaging or translation efficiency. In contrast, direct grafting shortens the encoded sequence and may improve expression, structural stability, and suitability for directed evolution. Comparing these two display formats is expected to clarify how surface engineering strategies influence the performance of receptor-targeted artificial nucleocapsids.
[Conclusion]
This study provides a foundation for the development of HER2-targeted artificial nucleocapsids as nonviral RNA delivery vehicles. By comparing two surface-display strategies, it offers insight into how exterior engineering influences the properties of receptor-targeted nucleocapsids. In the long term, if customizable RNA loading can be achieved, this platform may support the development of versatile and evolvable protein-based carriers for targeted delivery of diverse therapeutic RNAs.
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