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Nonviral ultrasound-mediated gene delivery in small and large animal models


Ultrasound-mediated gene delivery (sonoporation) is a minimally invasive, nonviral and clinically translatable method of gene therapy. This method offers a favorable safety profile over that of viral vectors and is less invasive as compared with other physical gene delivery approaches (e.g., electroporation). We have previously used sonoporation to overexpress transgenes in different skeletal tissues in order to induce tissue regeneration. Here, we provide a protocol that could easily be adapted to address various other targets of tissue regeneration or additional applications, such as cancer and neurodegenerative diseases. This protocol describes how to prepare, conduct and optimize ultrasound-mediated gene delivery in both a murine and a porcine animal model. The protocol includes the preparation of a microbubble–DNA mix and in vivo sonoporation under ultrasound imaging. Ultrasound-mediated gene delivery can be accomplished within 10 min. After DNA delivery, animals can be followed to monitor gene expression, protein secretion and other transgene-specific outcomes, including tissue regeneration. This procedure can be accomplished by a competent graduate student or technician with prior experience in ultrasound imaging or in performing in vivo procedures.

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Fig. 1: Spacer production.
Fig. 2: Sonoporation in mice.
Fig. 3: Sonoporation in pigs.
Fig. 4: Anticipated results.

Data availability

All materials are available from commercial sources or can be derived using methods described in this study. All relevant data are reported in the article.


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We acknowledge funding support from the California Institute for Regenerative Medicine (CIRM; (grant TR4-06713 to D.G.) and NIH grants R01EB026094 (to D.G.) and R01CA112356 (to K.W.F.).

Author information




G.P., K.W.F. and D.G. designed the experiments. M.B., J.F. and G.S. performed the experiments. M.B. and G.S. analyzed the data. M.B., J.F. and G.S. created the figures. M.B., J.F. and G.S. wrote the manuscript. All authors approved the manuscript.

Corresponding author

Correspondence to Dan Gazit.

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Competing interests

G.P. and D.G. are shareholders in GamlaStem Medical, which did not provide funds for this study. The other authors declare no competing interests.

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Shapiro, G. et al. J. Control. Release 223, 157–164 (2016):

Bez, M. et al. Sci. Transl. Med. 9, eaal3128 (2017):

Bez, M. et al. Mol. Ther. 26, 1746–1755 (2018):

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Bez, M., Foiret, J., Shapiro, G. et al. Nonviral ultrasound-mediated gene delivery in small and large animal models. Nat Protoc 14, 1015–1026 (2019).

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