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Noninvasive and targeted gene delivery into the brain using microbubble-facilitated focused ultrasound.

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.

SUBMITTER: Hsu PH

PROVIDER: S-EPMC3584045 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Noninvasive and targeted gene delivery into the brain using microbubble-facilitated focused ultrasound.

Hsu Po-Hung PH Wei Kuo-Chen KC Huang Chiung-Yin CY Wen Chih-Jen CJ Yen Tzu-Chen TC Liu Chao-Lin CL Lin Ya-Tin YT Chen Jin-Chung JC Shen Chia-Rui CR Liu Hao-Li HL

PloS one 20130227 2

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 controlle ...[more]

PMID: 23460893

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Project description:Focused ultrasound combined with microbubble-mediated intranasal delivery (FUSIN) is a new brain drug delivery technique. FUSIN utilizes the nasal route for direct nose-to-brain drug administration, thereby bypassing the blood-brain barrier (BBB) and minimizing systemic exposure. It also uses FUS-induced microbubble cavitation to enhance transport of intranasally (IN) administered agents to the FUS-targeted brain location. Previous studies have provided proof-of-concept data showing the feasibility of FUSIN to deliver dextran and the brain-derived neurotrophic factor to the caudate putamen of mouse brains. The objective of this study was to evaluate the biodistribution of IN administered gold nanoclusters (AuNCs) and assess the feasibility and short-term safety of FUSIN for the delivery of AuNCs to the brainstem. Three experiments were performed. First, the whole-body biodistribution of IN administered 64Cu-alloyed AuNCs (64Cu-AuNCs) was assessed using in vivo positron emission tomography/computed tomography (PET/CT) and verified with ex vivo gamma counting. Control mice were intravenously (IV) injected with the 64Cu-AuNCs. Second, 64Cu-AuNCs and Texas red-labeled AuNCs (TR-AuNCs) were used separately to evaluate FUSIN delivery outcome in the brain. 64Cu-AuNCs or TR-AuNCs were administered to mice through the nasal route, followed by FUS sonication at the brainstem in the presence of systemically injected microbubbles. The spatial distribution of 64Cu-AuNCs and TR-AuNCs were examined by autoradiography and fluorescence microscopy of ex vivo brain slices, respectively. Third, histological analysis was performed to evaluate any potential histological damage to the nose and brain after FUSIN treatment. The experimental results revealed that IN administration induced significantly lower 64Cu-AuNCs accumulation in the blood, lungs, liver, spleen, kidney, and heart compared with IV injection. FUSIN enhanced the delivery of 64Cu-AuNCs and TR-AuNCs at the FUS-targeted brain region compared with IN delivery alone. No histological-level tissue damage was detected in the nose, trigeminal nerve, and brain. These results suggest that FUSIN is a promising technique for noninvasive, spatially targeted, and safe delivery of nanoparticles to the brain with minimal systemic exposure.

Project description:BACKGROUND:The characteristic progression of Lewy pathology in Parkinson's disease likely involves intercellular exchange and the accumulation of misfolded ?-synuclein amplified by a prion-like self-templating mechanism. Silencing of the ?-synuclein gene could provide long-lasting disease-modifying benefits by reducing the requisite substrate for the spreading aggregation. OBJECTIVES:As a result of the poor penetration of viral vectors across the blood-brain barrier, gene therapy for central nervous system disorders requires direct injections into the affected brain regions, and invasiveness is further increased by the need for bilateral delivery to multiple brain regions. Here we test a noninvasive approach by combining low-intensity magnetic resonance-guided focused ultrasound and intravenous microbubbles that can transiently increase the access of brain impermeant therapeutic macromolecules to targeted brain regions. METHODS:Transgenic mice expressing human ?-synuclein were subjected to magnetic resonance-guided focused ultrasound targeted to 4 brain regions (hippocampus, substantia nigra, olfactory bulb, and dorsal motor nucleus) in tandem with intravenous microbubbles and an adeno-associated virus serotype 9 vector bearing a short hairpin RNA sequence targeting the ?-synuclein gene. RESULTS:One month following treatment, ?-synuclein immunoreactivity was decreased in targeted brain regions, whereas other neuronal markers such as synaptophysin or tyrosine hydroxylase were unchanged, and cell death and glial activation remained at basal levels. CONCLUSIONS:These results demonstrate that magnetic resonance-guided focused ultrasound can effectively, noninvasively, and simultaneously deliver viral vectors targeting ?-synuclein to multiple brain areas. Importantly, this approach may be useful to alter the progression of Lewy pathology along selected neuronal pathways, particularly as prodromal PD markers improve early diagnoses. © 2018 International Parkinson and Movement Disorder Society.

Project description:Müller glia are specialized retinal cells with stem cell properties in fish and frogs but not in mammals. Current efforts to develop gene therapies to activate mammalian Müller glia for retinal repair will require safe and effective delivery strategies for recombinant adeno-associated viruses (AAVs), vectors of choice for clinical translation. Intravitreal and subretinal injections are currently used for AAV gene delivery in the eye, but less invasive methods efficiently targeting Müller glia have yet to be developed. Methods: As gene delivery strategies have been more extensively studied in the brain, to validate our vectors, we initially compared the glial tropism of AAV-PHP.eB, an AAV9 that crosses the blood-brain and blood-retinal barriers, for its ability to drive fluorescent protein expression in glial cells in both the brain and retina. We then tested the glial transduction of AAV2/8-GFAP-mCherry, a virus that does not cross blood-brain and blood-retinal barriers, for its effectiveness in transducing Müller glia in murine retinal explants ex vivo. For in vivo assays we used larger rat eyes, performing invasive intravitreal injections, and non-invasive intravenous delivery using focused ultrasound (FUS) (pressure amplitude: 0.360 - 0.84 MPa) and microbubbles (Definity, 0.2 ml/kg). Results: We showed that AAV-PHP.eB carrying a ubiquitous promoter (CAG) and green fluorescent protein (GFP) reporter, readily crossed the blood-brain and blood-retinal barriers after intravenous delivery in mice. However, murine Müller glia did not express GFP, suggesting that they were not transduced by AAV-PHP.eB. We thus tested an AAV2/8 variant, which was selected based on its safety record in multiple clinical trials, adding a glial fibrillary acidic protein (GFAP) promoter and mCherry (red fluorescent protein) reporter. We confirmed the glial specificity of AAV2/8-GFAP-mCherry, showing effective expression of mCherry in astrocytes after intracranial injection in the mouse brain, and of Müller glia in murine retinal explants. For in vivo experiments we switched to rats because of their larger size, injecting AAV2/8-GFAP-mCherry intravitreally, an invasive procedure, demonstrating passage across the inner limiting membrane, leading to Müller glia transduction. We then tested an alternative non-invasive delivery approach targeting a different barrier - the inner blood-retinal-barrier, applying focused ultrasound (FUS) to the retina after intravenous injection of AAV2/8 and microbubbles in rats, using magnetic resonance imaging (MRI) for FUS targeting. FUS permeabilized the rat blood-retinal-barrier and allowed the passage of macromolecules to the retina (Evans blue, IgG, IgM), with minimal extravasation of platelets and red blood cells. Intravenous injection of microbubbles and AAV2/8-GFAP-mCherry followed by FUS resulted in mCherry expression in rat Müller glia. However, systemic delivery of AAV2/8 also had off-target effects, transducing several murine peripheral organs, particularly the liver. Conclusions: Retinal permeabilisation via FUS in the presence of microbubbles is effective for delivering AAV2/8 across the inner blood-retinal-barrier, targeting Müller glia, which is less invasive than intravitreal injections that bypass the inner limiting membrane. However, implementing FUS in the clinic will require a comprehensive consideration of any off-target tropism of the AAV in peripheral organs, combined ideally, with the development of Müller glia-specific promoters.

Dataset Information

Noninvasive and targeted gene delivery into the brain using microbubble-facilitated focused ultrasound.

Publications

Noninvasive and targeted gene delivery into the brain using microbubble-facilitated focused ultrasound.

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