Non-invasive, neuron-specific gene therapy by focused ultrasound-induced blood-brain barrier opening in Parkinson's disease mouse model.

Focused ultrasound (FUS)-induced with microbubbles (MBs) is a promising technique for noninvasive opening of the blood-brain barrier (BBB) to allow targeted delivery of therapeutic substances into the brain and thus the noninvasive delivery of gene vectors for CNS treatment. We have previously demonstrated that a separated gene-carrying liposome and MBs administration plus FUS exposure can deliver genes into the brain, with the successful expression of the reporter gene and glial cell line-derived neurotrophic factor (GDNF) gene. In this study, we further modify the delivery system by conjugating gene-carrying liposomes with MBs to improve the GDNF gene-delivery efficiency, and to verify the possibility of using this system to perform treatment in the 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced animal disease model. FUS-BBB opening was verified by contrast-enhanced MRI, and GFP gene expression was verified via in vivo imaging system (IVIS). Western blots as well as enzyme-linked immunosorbent assay (ELISA) were conducted to measure protein expression, and immunohistochemistry (IHC) was conducted to test the Tyrosine hydroxylase (TH)-neuron distribution. Dopamine (DA) and its metabolites as well as dopamine active transporter (DAT) were quantitatively analyzed to show dopaminergic neuronal dopamine secretion/activity/metabolism. Motor performance was evaluated by rotarod test weekly. Results demonstrated that the LpDNA-MBs (gene-liposome-MBs) complexes successfully serve as gene carrier and BBB-opening catalyst, and outperformed the separated LpDNA/MBs administration both in terms of gene delivery and expression. TH-positive IHC and measurement of DA and its metabolites DOPAC and HVA confirmed improved neuronal function, and the proposed system also provided the best neuroprotective effect to retard the progression of motor-related behavioral abnormalities. Immunoblotting and histological staining further confirmed the expression of reporter genes in neuronal cells. This study suggests that FUS exposures with the administration of LpDNA-MBs complexes synergistically can serve as an effective gene therapy strategy for MPTP-animal treatment, and may have potential for further application to perform gene therapy for neurodegenerative disease.

Focused ultrasound-induced blood-brain barrier opening for non-viral, non-invasive, and targeted gene delivery.

Focused ultrasound (FUS) exposure in the presence of microbubbles can temporally open the blood-brain barrier (BBB) and is an emerging technique for non-invasive brain therapeutic agent delivery. Given the potential to deliver large molecules into the CNS via this technique, we propose a reliable strategy to synergistically apply FUS-BBB opening for the non-invasive and targeted delivery of non-viral genes into the CNS for therapeutic purpose. In this study, we developed a gene-liposome system, in which the liposomes are designed to carry plasmid DNA (pDNA, containing luciferase reporter gene) to form a liposomal-plasmid DNA (LpDNA) complex. Pulsed FUS exposure was delivered to induce BBB opening (500-kHz, burst length=10ms, 1% duty cycle, PRF=1Hz). The longitudinal expression of luciferase was quantitated via an in vivo imaging system (IVIS). The reporter gene expression level was confirmed via immunoblotting, and histological staining was used to identify transfected cells via fluorescent microscopy. In a comparison of gene transduction efficiency, the LpDNA system showed better cell transduction than the pDNA system. With longitudinal observation of IVIS monitoring, animals with FUS treatment showed significant promotion of LpDNA release into the CNS and demonstrated enhanced expression of genes upon sonication with FUS-BBB opening, while both the luciferase and GDNF protein expression were successfully measured via Western blotting. The gene expression peak was observed at day 2, and the gene expression level was up to 5-fold higher than that in the untreated hemisphere (compared to a 1-fold increase in the direct-inject positive-control group). The transfection efficiency was also found to be LpDNA dose-dependent, where higher payloads of pDNA resulted in a higher transfection rate. Immunoblotting and histological staining confirmed the expression of reporter genes in glial cells as well as astrocytes. This study suggests that IV administration of LpDNA in combination with FUS-BBB opening can provide effective gene delivery and expression in the CNS, demonstrating the potential to achieve non-invasive and targeted gene delivery for treatment of CNS diseases.