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

Abstract

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1.

    Martinek, V. et al. Genetic engineering of meniscal allografts. Tissue Eng. 8, 107–117 (2002).

    CAS  Article  Google Scholar 

  2. 2.

    Jacobson, S. G. et al. Improvement and decline in vision with gene therapy in childhood blindness. N. Engl. J. Med. 372, 1920–1926 (2015).

    CAS  Article  Google Scholar 

  3. 3.

    Pierce, G. F., Lillicrap, D., Pipe, S. W. & Vandendriessche, T. Gene therapy, bioengineered clotting factors and novel technologies for hemophilia treatment. J. Thromb. Haemost. 5, 901–906 (2007).

    CAS  Article  Google Scholar 

  4. 4.

    Wang, Z. G., Wu, Z. Q., Liu, Y. & Han, W. D. New development in CAR-T cell therapy. J. Hematol. Oncol. 10, 53 (2017).

  5. 5.

    Weber, T. Do we need marker gene studies in humans to improve clinical AAV gene therapy? Gene Ther. 24, 72–73 (2017).

    CAS  Article  Google Scholar 

  6. 6.

    Sheyn, D. et al. Genetically modified cells in regenerative medicine and tissue engineering. Adv. Drug Deliv. Rev. 62, 683–698 (2010).

    CAS  Article  Google Scholar 

  7. 7.

    Kimelman Bleich, N. et al. Gene therapy approaches to regenerating bone. Adv. Drug Deliv. Rev. 64, 1320–1330 (2012).

    CAS  Article  Google Scholar 

  8. 8.

    Pelled, G. et al. Direct gene therapy for bone regeneration: gene delivery, animal models, and outcome measures. Tissue Eng. Part B Rev. 16, 13–20 (2010).

    CAS  Article  Google Scholar 

  9. 9.

    Bessis, N., GarciaCozar, F. J. & Boissier, M. C. Immune responses to gene therapy vectors: influence on vector function and effector mechanisms. Gene Ther. 11, S10–S17 (2004). (Suppl 1).

    CAS  Article  Google Scholar 

  10. 10.

    Baum, C., Kustikova, O., Modlich, U., Li, Z. & Fehse, B. Mutagenesis and oncogenesis by chromosomal insertion of gene transfer vectors. Hum. Gene Ther. 17, 253–263 (2006).

    CAS  Article  Google Scholar 

  11. 11.

    Yoon, C. S. & Park, J. H. Ultrasound-mediated gene delivery. Expert Opin. Drug Deliv. 7, 321–330 (2010).

    CAS  Article  Google Scholar 

  12. 12.

    Sheyn, D. et al. Ultrasound-based nonviral gene delivery induces bone formation in vivo. Gene Ther. 15, 257–266 (2008).

    CAS  Article  Google Scholar 

  13. 13.

    Marrero, B., Shirley, S. & Heller, R. Delivery of interleukin-15 to B16 melanoma by electroporation leads to tumor regression and long-term survival. Technol. Cancer Res. Treat. 13, 551–560 (2014).

    PubMed  PubMed Central  Google Scholar 

  14. 14.

    Ayuni, E. L. et al. In vivo electroporation mediated gene delivery to the beating heart. PLoS ONE 5, e14467 (2010).

    Article  Google Scholar 

  15. 15.

    Ferrara, K. W. Driving delivery vehicles with ultrasound. Adv. Drug Deliv. Rev. 60, 1097–1102 (2008).

    CAS  Article  Google Scholar 

  16. 16.

    Sanches, P. G. et al. Ultrasound-mediated gene delivery of naked plasmid DNA in skeletal muscles: a case for bolus injections. J Control. Release 195, 130–137 (2014).

    CAS  Article  Google Scholar 

  17. 17.

    Watanabe, Y. et al. Delivery of Na/I symporter gene into skeletal muscle using nanobubbles and ultrasound: visualization of gene expression by PET. J. Nucl. Med. 51, 951–958 (2010).

    CAS  Article  Google Scholar 

  18. 18.

    Li, T., Tachibana, K., Kuroki, M. & Kuroki, M. Gene transfer with echo-enhanced contrast agents: comparison between Albunex, Optison, and Levovist in mice—initial results. Radiology 229, 423–428 (2003).

  19. 19.

    Lu, Q. L., Liang, H. D., Partridge, T. & Blomley, M. J. Microbubble ultrasound improves the efficiency of gene transduction in skeletal muscle in vivo with reduced tissue damage. Gene Ther. 10, 396–405 (2003).

    CAS  Article  Google Scholar 

  20. 20.

    Wang, X., Liang, H. D., Dong, B., Lu, Q. L. & Blomley, M. J. Gene transfer with microbubble ultrasound and plasmid DNA into skeletal muscle of mice: comparison between commercially available microbubble contrast agents. Radiology 237, 224–229 (2005).

    Article  Google Scholar 

  21. 21.

    Qin, S., Caskey, C. F. & Ferrara, K. W. Ultrasound contrast microbubbles in imaging and therapy: physical principles and engineering. Phys. Med. Biol. 54, R27–R57 (2009).

    Article  Google Scholar 

  22. 22.

    van Wamel, A., Bouakaz, A., Bernard, B., ten Cate, F. & de Jong, N. Radionuclide tumour therapy with ultrasound contrast microbubbles. Ultrasonics 42, 903–906 (2004).

    Article  Google Scholar 

  23. 23.

    De Cock, I. et al. Ultrasound and microbubble mediated drug delivery: acoustic pressure as determinant for uptake via membrane pores or endocytosis. J. Control. Release 197, 20–28 (2015).

    Article  Google Scholar 

  24. 24.

    Kimelman-Bleich, N. et al. Targeted gene-and-host progenitor cell therapy for nonunion bone fracture repair. Mol. Ther. 19, 53–59 (2011).

    CAS  Article  Google Scholar 

  25. 25.

    Shapiro, G. et al. Ultrasound-mediated transgene expression in endogenous stem cells recruited to bone injury sites. Polym. Adv. Technol. 25, 525–531 (2014).

    CAS  Article  Google Scholar 

  26. 26.

    Shapiro, G. et al. Multiparameter evaluation of in vivo gene delivery using ultrasound-guided, microbubble-enhanced sonoporation. J. Control. Release 223, 157–164 (2016).

    CAS  Article  Google Scholar 

  27. 27.

    Bez, M. et al. In situ bone tissue engineering via ultrasound-mediated gene delivery to endogenous progenitor cells in mini-pigs. Sci. Transl. Med. 9, eaal3128 (2017).

  28. 28.

    Bez, M. et al. Ultrasound-mediated gene delivery enhances tendon allograft integration in mini-pig ligament reconstruction. Mol. Ther. 26, 1746-1755 (2018).

  29. 29.

    Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M. & Altman, D. G. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol. 8, e1000412 (2010).

    Article  Google Scholar 

  30. 30.

    Fan, Z., Kumon, R. E. & Deng, C. X. Mechanisms of microbubble-facilitated sonoporation for drug and gene delivery. Ther. Deliv. 5, 467–486 (2014).

    CAS  Article  Google Scholar 

  31. 31.

    Delalande, A. et al. Cationic gas-filled microbubbles for ultrasound-based nucleic acids delivery. Biosci. Rep. 37, BSR20160619 (2017).

  32. 32.

    Sheyn, D. et al. PTH induces systemically administered mesenchymal stem cells to migrate to and regenerate spine injuries. Mol. Ther. 24, 318–330 (2016).

    CAS  Article  Google Scholar 

  33. 33.

    Zachos, T. A., Shields, K. M. & Bertone, A. L. Gene-mediated osteogenic differentiation of stem cells by bone morphogenetic proteins-2 or -6. J. Orthop. Res. 24, 1279–1291 (2006).

    CAS  Article  Google Scholar 

  34. 34.

    Lai, C. Y. et al. Noninvasive thermometry assisted by a dual-function ultrasound transducer for mild hyperthermia. IEEE Trans. Ultrason. Ferroelectr. Freq. Control 57, 2671–2684 (2010).

    Article  Google Scholar 

  35. 35.

    Kheirolomoom, A. et al. Complete regression of local cancer using temperature-sensitive liposomes combined with ultrasound-mediated hyperthermia. J. Control. Release 172, 266–273 (2013).

    CAS  Article  Google Scholar 

  36. 36.

    Jingfei, L., Josquin, F., Douglas, N. S., Olivier Le, B. & Katherine, W. F. Development of a spherically focused phased array transducer for ultrasonic image-guided hyperthermia. Phys. Med. Biol. 61, 5275–5296 (2016).

    Article  Google Scholar 

  37. 37.

    Shapiro, G. et al. Multiparameter evaluation of in vivo gene delivery using ultrasound-guided, microbubble-enhanced sonoporation. J. Control. Release 223, 157–164 (2016).

    CAS  Article  Google Scholar 

  38. 38.

    Fujii, H. et al. Repeated and targeted transfer of angiogenic plasmids into the infarcted rat heart via ultrasound targeted microbubble destruction enhances cardiac repair. Eur. Heart J. 32, 2075–2084 (2011).

    CAS  Article  Google Scholar 

  39. 39.

    Leong-Poi, H. et al. Therapeutic arteriogenesis by ultrasound-mediated VEGF165 plasmid gene delivery to chronically ischemic skeletal muscle. Circ. Res. 101, 295–303 (2007).

    CAS  Article  Google Scholar 

  40. 40.

    Zolochevska, O. et al. Sonoporation delivery of interleukin-27 gene therapy efficiently reduces prostate tumor cell growth in vivo. Hum. Gene Ther. 22, 1537–1550 (2011).

    CAS  Article  Google Scholar 

  41. 41.

    Hauff, P. et al. Evaluation of gas-filled microparticles and sonoporation as gene delivery system: feasibility study in rodent tumor models. Radiology 236, 572–578 (2005).

    Article  Google Scholar 

  42. 42.

    Shimamura, M. et al. Development of efficient plasmid DNA transfer into adult rat central nervous system using microbubble-enhanced ultrasound. Gene Ther. 11, 1532–1539 (2004).

    CAS  Article  Google Scholar 

  43. 43.

    Takeuchi, D. et al. Alleviation of Abeta-induced cognitive impairment by ultrasound-mediated gene transfer of HGF in a mouse model. Gene Ther. 15, 561–571 (2008).

    CAS  Article  Google Scholar 

  44. 44.

    Nakashima, M. et al. Induction of reparative dentin formation by ultrasound-mediated gene delivery of growth/differentiation factor 11. Hum. Gene Ther. 14, 591–597 (2003).

    CAS  Article  Google Scholar 

  45. 45.

    Anderson, C. D., Moisyadi, S., Avelar, A., Walton, C. B. & Shohet, R. V. Ultrasound-targeted hepatic delivery of factor IX in hemophiliac mice. Gene Ther. 23, 510–519 (2016).

    CAS  Article  Google Scholar 

  46. 46.

    Chen, S. et al. Efficient gene delivery to pancreatic islets with ultrasonic microbubble destruction technology. Proc. Natl. Acad. Sci. USA 103, 8469–8474 (2006).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

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

Affiliations

Authors

Contributions

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.

Ethics declarations

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.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Shapiro, G. et al. J. Control. Release 223, 157–164 (2016): https://doi.org/10.1016/j.jconrel.2015.12.001

Bez, M. et al. Sci. Transl. Med. 9, eaal3128 (2017): https://doi.org/10.1126/scitranslmed.aal3128

Bez, M. et al. Mol. Ther. 26, 1746–1755 (2018): https://doi.org/10.1016/j.ymthe.2018.04.020

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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). https://doi.org/10.1038/s41596-019-0125-y

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.