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.