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Sonoselective delivery using ultrasound and microbubbles combined with intravenous rAAV9 CLDN5-GFP does not increase endothelial gene expression

Gene Therapy (2023)Cite this article

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Abstract

Transcranial ultrasound combined with intravenous microbubbles can be used to increase blood-brain barrier permeability or, at lower pressures, to mediate sonoselective gene delivery to endothelial cells. Previously, sonoselective gene delivery with plasmid-coated microbubbles as gene carriers resulted in transient transgene expression in the brain endothelium. We investigated the potential of recombinant adeno-associated virus 9 (rAAV9), a serotype known for its efficient transduction and long-term transgene expression, for sonoselective gene delivery to endothelial cells of the brain. We found that rAAV9 led to gene delivery to brain endothelial cells following intravenous administration at a dosage of 1 × 1011 GC/g. However, the sonoselective gene delivery approach with intravenous rAAV9, using the same parameters as previously used for plasmid delivery, did not increase transgene expression in brain endothelial cells targeted. These results suggest that intravenous rAAV9 are using mechanisms of entry into the cerebrovasculature that are not significantly influenced by sonoselective treatments known to facilitate endothelial cell entry of plasmids coated onto microbubbles.

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Fig. 1: Pilot Study: CLDN5 promoter provides high GFP expression in brain vasculature.
Fig. 2: Ultrasound treatment and rAAV administration.
Fig. 3: Sonoselective modulation of the BBB does not enhance gene delivery by intravenous rAAV9 CLDN5-GFP to endothelium.
Fig. 4: Schematic representation of the hypothesis.

Data availability

Data is available from corresponding authors on reasonable request. Viral genome plasmids pEMS2143 and pEMS2181 are available to the research community through the nonprofit distributor Addgene (Cambridge, MA) (www.addgene.org).

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Acknowledgements

The authors thank Kristina Mikloska and Shawna Rideout for their invaluable contributions during focused ultrasound treatments. For imaging we are grateful to Microscopy and Imaging Laboratory (MIL) at the University of Toronto and Centre for Flow Cytometry and Microscopy at Sunnybrook Research Institute. We thank Dr. Yutaka Amemiya at the Genomics Core Facility, Sunnybrook Research Institute, for droplet digital PCR analysis. Salary support was provided by Carlsberg Internationalisation Fellowship (#CF20-0379 to RHK) and Canada Research Chairs Program (CRC Tier 1 in Brain Repair and Regeneration to IA). This research was supported by Canadian Institute of Health Research (to IA: 137064, 166184, and 168906; and to KH: 154272), the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health (KH: RO1-EB003268), the Temerty Chair in Focused Ultrasound Research (KH), and FDC Foundation, the WB Family Foundation, Gerald and Carla Connor. EMS received funding from the Weston Brain Institute (TR160014).

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Authors and Affiliations

  1. Biological Sciences, Hurvitz Brain Sciences Research Program, Sunnybrook Research Institute, Toronto, ON, Canada

    Rikke Hahn Kofoed & Isabelle Aubert

  2. Department of Laboratory Medicine and Pathobiology, Temerty Faculty of Medicine, University of Toronto, Toronto, ON, Canada

    Rikke Hahn Kofoed & Isabelle Aubert

  3. Centre for Molecular Medicine and Therapeutics at British Columbia Children’s Hospital, Department of Medical Genetics, The University of British Columbia, Vancouver, Canada

    Elizabeth M. Simpson

  4. Physical Sciences, Sunnybrook Research Institute, Toronto, ON, Canada

    Kullervo Hynynen

  5. Department of Medical Biophysics, Temerty Faculty of Medicine, University of Toronto, Toronto, ON, Canada

    Kullervo Hynynen

Authors
  1. Rikke Hahn Kofoed
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  4. Isabelle Aubert
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Contributions

RHK, IA, and KH designed the study. EMS designed and supplied rAAV vectors. RHK performed experiments, tissue processing, data analysis, and created figures and manuscript drafts. All authors edited and proofread the manuscript.

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Correspondence to Rikke Hahn Kofoed or Isabelle Aubert.

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Competing interests

RHK, KH, and IA declare no competing interest. EMS holds issued patents on CLDN5 MiniPromoters.

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Kofoed, R.H., Simpson, E.M., Hynynen, K. et al. Sonoselective delivery using ultrasound and microbubbles combined with intravenous rAAV9 CLDN5-GFP does not increase endothelial gene expression. Gene Ther (2023). https://doi.org/10.1038/s41434-023-00389-y

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