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Cited by 136Ultrasound and Hyperthermia ApplicationsVirus-based gene therapy researchBarrier Structure and Function Studies

The DOI matches its Crossref record; 56 of the 57 references it cites were found in a registry or an index.

Noninvasive and Targeted Gene Delivery into the Brain Using Microbubble-Facilitated Focused Ultrasound

Open access
Hsu, Po-Hung, Wei, Kuo-Chen, Huang, Chiung-Yin, Wen, Chih-Jen, Yen, Tzu-Chen, Liu, Chao-Lin, Lin, Ya-Tin, Chen, Jin-Chung, Shen, Chia-Rui, Liu, Hao-Li·2013·PLoS ONE
DOI 10.1371/journal.pone.0057682PMC PMC3584045
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Abstract

Recombinant adeno-associated viral (rAAV) vectors are potentially powerful tools for gene therapy of CNS diseases, but their penetration into brain parenchyma is severely limited by the blood-brain barrier (BBB) and current delivery relies on invasive stereotactic injection. Here we evaluate the local, targeted delivery of rAAV vectors into the brains of mice by noninvasive, reversible, microbubble-facilitated focused ultrasound (FUS), resulting in BBB opening that can be monitored and controlled by magnetic resonance imaging (MRI). Using this method, we found that IV-administered AAV2-GFP (green fluorescence protein) with a low viral vector titer (1×10(9) vg/g) can successfully penetrate the BBB-opened brain regions to express GFP. We show that MRI monitoring of BBB-opening could serve as an indicator of the scale and distribution of AAV transduction. Transduction peaked at 3 weeks and neurons and astrocytes were affected. This novel, noninvasive delivery approach could significantly broaden the application of AAV-viral-vector-based genes for treatment of CNS diseases.

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Figure 1 Scheme of the study. AAV2 harboring a specific gene (hrGFP) is IV-injected into animals. Delivery of focused ultrasound to the animal brain causes virus to leak into the brain through the opened BBB, where it transfects cells. Transfected cells will produce cytoplasmic GFP that can be detected by immunofluoresence microscopy of mouse brains.
Figure 1 Scheme of the study. AAV2 harboring a specific gene (hrGFP) is IV-injected into animals. Delivery of focused ultrasound to the animal brain causes virus to leak into the brain through the opened BBB, where it transfects cells. Transfected cells will produce cytoplasmic GFP that can be detected by immunofluoresence microscopy of mouse brains.CC BY · PubMed Central
Figure 2 Correlation between MRI and GFP expressions. (a) T1-weighted MR images showing BBB disruption by different acoustic pressures (bar = 1 mm) and (b) respective GFP expression by immunofluorescence (bar = 200 µm). (c) Increase in GFP signal intensity at different acoustic powers. (d) Correlation of the change in MR signal intensity with the increase in AAV2-GFP expression (r2 = 0.687). In (a), circles in dashed lines represent the FUS exposure area, and the rectangular regions represents the zoomed immunofluorescence regions.
Figure 2 Correlation between MRI and GFP expressions. (a) T1-weighted MR images showing BBB disruption by different acoustic pressures (bar = 1 mm) and (b) respective GFP expression by immunofluorescence (bar = 200 µm). (c) Increase in GFP signal intensity at different acoustic powers. (d) Correlation of the change in MR signal intensity with the increase in AAV2-GFP expression (r2 = 0.687). In (a), circles in dashed lines represent the FUS exposure area, and the rectangular regions represents the zoomed immunofluorescence regions.CC BY · PubMed Central
Figure 3 Longitudinal GFP-expression profiles. (a) GFP expression assayed weekly from week 1 (D7) to week 6 (D42), and in negative and positive controls. Top panels, sonicated brain; lower panels, contralateral control. Bar = 100 µm. (b) Evaluation of increase in GFP signal intensity at different times after transfection.
Figure 3 Longitudinal GFP-expression profiles. (a) GFP expression assayed weekly from week 1 (D7) to week 6 (D42), and in negative and positive controls. Top panels, sonicated brain; lower panels, contralateral control. Bar = 100 µm. (b) Evaluation of increase in GFP signal intensity at different times after transfection.CC BY · PubMed Central
Figure 4 Confirmation of GFP expression by immunofluorescence and Western blot (3 weeks after AAV transduction). (a) AAV2-GFP expression as detected by immunofluorescence of GFP; (b) anti-GFP rhodamine immunofluorescence; (c) overlaid GFP expression and anti-GFP rhodamine to demonstrate their colocalization; (d) Western blot of β-tubulin and GFP expression in control and FUS-exposed brains, demonstrating that GFP was only expressed in the experimental group. Bar = 100 µm.
Figure 4 Confirmation of GFP expression by immunofluorescence and Western blot (3 weeks after AAV transduction). (a) AAV2-GFP expression as detected by immunofluorescence of GFP; (b) anti-GFP rhodamine immunofluorescence; (c) overlaid GFP expression and anti-GFP rhodamine to demonstrate their colocalization; (d) Western blot of β-tubulin and GFP expression in control and FUS-exposed brains, demonstrating that GFP was only expressed in the experimental group. Bar = 100 µm.CC BY · PubMed Central
Figure 5 Colocalization of AAV-infected area with other markers (3 weeks after AAV transduction). (a) GFP (green) and neuronal nuclei (NeuN, red) staining, bar = 10 µm.; (b) GFP (green) and GFAP (red) colocalization by fluorescence microscopy, bar = 10 µm.
Figure 5 Colocalization of AAV-infected area with other markers (3 weeks after AAV transduction). (a) GFP (green) and neuronal nuclei (NeuN, red) staining, bar = 10 µm.; (b) GFP (green) and GFAP (red) colocalization by fluorescence microscopy, bar = 10 µm.CC BY · PubMed Central

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Research works printed but not linked: 1

  1. N Carty, 2010, Convection-enhanced delivery and systemic mannitol increase gene product distribution of AAV vectors 5, 8, and 9 and increase gene product in the adult mouse brain, J Neurosci Methods, 194, 144, 10.1016/j.jneumeth.2010.10.010 · DOI

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Metadata

Authors
Hsu, P.-H., Wei, K.-C., Huang, C.-Y., Wen, C.-J., Yen, T.-C., Liu, C.-L., Lin, Y.-T., Chen, J.-C., Shen, C.-R., & Liu, H.-L.
Affiliations
  • Chang Gung University (TW)
  • Ming Chi University of Technology (TW)
Corresponding author
  • Shen, Chia-Rui
  • Liu, Hao-Li
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2013-02-27
Volume
8
Issue
2
Pages
e57682
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0057682
PMC
PMC3584045
OpenAlex
W1969401867
Cited by
136
References
57
Citations by year
2026: 5 · 2025: 8 · 2024: 9 · 2023: 4 · 2022: 12 · 2021: 20 · 2020: 12 · 2019: 12 · 2018: 12 · 2017: 3 · 2016: 9 · 2015: 14 · 2014: 13 · 2013: 2
FWCI
6.99
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • National Science Council (100-2321-B-182-010)
  • Chang Gung Medical Foundation
Topics
  • Physical Sciences › Engineering › Biomedical Engineering › Ultrasound and Hyperthermia Applications
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
  • Life Sciences › Neuroscience › Neurology › Barrier Structure and Function Studies
Keywords
gene therapy, adeno-associated virus vectors, focused ultrasound, microbubbles, blood-brain barrier disruption, central nervous system disorders
MeSH terms
Dependovirus, Animals, Astrocytes, Blood-Brain Barrier, Brain, Contrast Media, Fluorescent Antibody Technique, Genetic Vectors, Magnetic Resonance Imaging, Male, Mice, Inbred ICR, Transduction, Genetic, Ultrasonics, Blotting, Western, Reproducibility of Results, Gene Transfer Techniques, Microbubbles, Green Fluorescent Proteins, Mice

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