Enhanced mRNA delivery using ultrasound-delivered click reactive anchors

Di Ianni, E.; Jeon, J.; Hu, H.; Quintana, J. M.; Lee, C.; Haidar, E. A.; Mohammadi, S.; Zargani-Piccardi, A.; Mahamdeh, M.; Hernandez, I. C.; Ng, T. S. C.; Breyne, K.; Lee, H.; Breakefield, X. O.; Miller, M. A.

doi:10.1101/2025.01.28.635330

bioRxiv · 10.1101/2025.01.28.635330

Enhanced mRNA delivery using ultrasound-delivered click reactive anchors

Di Ianni, E.·Jeon, J.·Hu, H.·Quintana, J. M.·Lee, C.·Haidar, E. A.·Mohammadi, S.·Zargani-Piccardi, A.·Mahamdeh, M.·Hernandez, I. C.·Ng, T. S. C.·Breyne, K.·Lee, H.·Breakefield, X. O.·Miller, M. A.

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Abstract

Therapeutic nucleic acid delivery has many potential applications, but it remains challenging to target extrahepatic tissues in a flexible and image-guided manner. To address this issue, we report a bioorthogonal pre-targeting strategy that uses focused ultrasound to promote the delivery of mRNA-loaded lipid nanoparticles (mRNA-LNP). We synthesized amphiphilic click reactive anchors (ACRAs) consisting of a phospholipid PEG-conjugate functionalized with transcyclooctene (TCO) or its companion reactive partner methyltetrazine (mTz), yielding ACRA-TCO and ACRA-mTz. ACRA derivatives were screened for cellular activity, yielding functionalized DOPE-PEG (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- (polyethylene glycol)) derivatives outperforming those containing saturated lipid or branched PEG. Nanobubbles encapsulating ultrasound-responsive gas precursor delivered ACRA-TCO to targeted cells and tissues using focused ultrasound, and this pre-targeting promoted the subsequent delivery of mRNA- LNP functionalized with companion ACRA-mTz. In cell cultures and in mice, ultrasound pre-targeting enhanced the accumulation of mTz-functionalized small molecule and nanoparticle compounds by 75% and 3.6-fold, respectively, and increased gene expression using mRNA-LNP in vivo. Taken together, this report presents a modular, ultrasound-enabled strategy for enhancing nucleic acid delivery in targeted tissues.

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Di Ianni, E., Jeon, J., Hu, H., Quintana, J. M., Lee, C., Haidar, E. A., Mohammadi, S., Zargani-Piccardi, A., Mahamdeh, M., Hernandez, I. C., Ng, T. S. C., Breyne, K., Lee, H., Breakefield, X. O., Miller, M. A.. 2025-01-28. Enhanced mRNA delivery using ultrasound-delivered click reactive anchors. https://doi.org/10.1101/2025.01.28.635330

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