Abstract
Gene therapy in the central nervous system (CNS) has the potential to slow or reverse pathology in numerous neurological diseases including Parkinson’s disease, Alzheimer’s disease, and brain tumors. However, while clinical gene therapy trials have been largely unsuccessful, outcomes may be improved by a) enhancing delivery efficiency and transfection volume, b) improving reproducibility of treatments and c) treating patients at earlier or prodromal stages prior to onset of irreversible pathology. While currently the gold standard for treatments in the CNS, direct administration strategies are invasive and may yield poor gene vector distribution. Less invasive strategies capable of targeted and homogenous delivery in the CNS are required.
In this dissertation, we demonstrate a novel strategy for delivery of gene vector nanoparticles into the brain capable of circumventing two major barriers to drug delivery in the brain; namely, the blood-brain barrier (BBB) and the nanoporous extracellular matrix (ECM). Focused ultrasound (FUS), when used in conjunction with ultrasound contrast agent microbubbles (MBs) is capable of non-invasive and spatially localized disruption of the BBB, capable of delivering agents as large as 100 nm in diameter into the CNS. We use this strategy in combination with non-viral gene vector nanoparticles which are coated in exceptionally dense coats of polyethylene glycol in a “brain-penetrating” nanoparticle (BPN) formulation, capable of rapidly diffusing through brain tissue. With this novel strategy, we are able to achieve robust reporter gene expression in the brain of healthy rats that lasted at least 28-days and was localized to the site targeted with FUS. Importantly, we demonstrate transfection of both neurons and astrocytes without signs of toxicity or astrocyte activation.
Next, we sought to apply our strategy to a rat model of Parkinson’s disease (PD). PD is a common and idiopathic neurodegenerative disorder commonly characterized by degeneration of dopamine-generating neurons in the substantia nigra and their axonal projections into the striatum. We packaged a gene for the glial cell line-derived neurotrophic factor (GDNF), a neurotrophic factor shown by other groups to be therapeutic in this model, into a BPN and delivered it to the striatum of a PD rat model. With just a single treatment, we were able to achieve therapeutically relevant levels of GDNF protein content in the FUS-targeted striatum, restore dopamine levels, and dopaminergic neuron density and eliminate behavioral indicators of Parkinsonism.
Finally, we explored strategies to enhance BPN dispersion and uptake in the CNS. By using a unique approach in which pulsed FUS was used to pre-treat the target tissue, we found significant enhancement in BPN dispersion through healthy rat and mouse brain as well as in the U87 model of human glioblastoma multiforme after infusion into the brain parenchyma with convection enhanced delivery. Next, by applying this pre-treatment regimen prior to opening of the BBB with FUS and MBs, we demonstrate up to 5-fold enhancement of delivery of BPN compared animals whose BBB was opened with FUS and MBs but did not receive pre-treatment FUS.
Overall, these studies demonstrate the ability of FUS to target the delivery of gene vectors across the BBB and elicit robust and homogenous transgene expression that is localized to the FUS-targeted site. Importantly, the non-invasive nature allows treatment of neurological disorders earlier time points than current invasive direct administration strategies. These results will be important in the development and, ultimately, translation of FUS-based strategies for gene delivery and gene therapy in the CNS.
