(725f) Dual-Responsive Polymersomes As In Vivo Enzyme Replacement Therapy to the Brain

Approximately 1 in 5,000 to 8,000 children are born annually with a lysosomal storage disease (LSD) in which one or more enzymes necessary for cellular function are not produced, leading to early and high mortality of those affected. Current treatment of LSDs, called enzyme replacement therapy (ERT), involves frequent intravenous (IV) infusions of missing endogenous enzymes. While ERT has proven effective in LSDs without central nervous system (CNS) involvement, the brain has remained untreatable due to the presence of the blood-brain barrier (BBB), which prevents passage of 98% of small molecule drugs, including enzymes, from the blood into the brain. Patients with GM1 gangliosidosis, a neuropathic LSD, do not produce the lysosomal enzyme β-galactosidase (βgal) and present with severe CNS degeneration, ataxia, and premature death, with no treatment available. Encapsulating enzyme into polymersomes facilitates the transport of this missing enzyme to the brain of GM1 gangliosidosis affected felines through IV injections, effectively extending ERT to the brain for the first time.

Polymersomes formed using poly(ethylene glycol)-b- poly(lactic acid) (PEGPLA) can encapsulate, protect, and deliver βgal. PEGPLA polymersomes form via solvent injection with an average diameter of 145 ± 21 nm, encapsulate βgal at 72.0 ± 12.2% efficiency and demonstrate simultaneous encapsulation and ligand attachment at 86.7 ± 11.6% efficiency. Amine-reactive PEG facilitated the attachment of apolipoprotein E (ApoE), a target to the low density lipoprotein family of receptors for BBB delivery, to the polymersome surface. In vitro, PEGPLA polymersomes demonstrate limited release in physiologic environment, pH 7.4, with a burst release upon membrane poration in lysosomal environment, pH 4.8, which is as desired for delivery of βgal to the lysosome. Cellular studies, using GM1 gangliosidosis-diseased fibroblasts, confirm that βgal-loaded polymersomes increase enzyme activity to normal levels with doses as low as 0.7 mg/cm². The addition of ApoE as a targeting ligand decreases the required dose to 0.175 mg/cm².

Finally, four-week-old GM1 gangliosidosis felines were injected with 760 mg/kg ApoE-tagged polymersomes via intravenous catheter. After 24 hours, cerebrospinal fluid βgal activity was elevated approximately seven-fold over age-match controlled untreated animals, indicating passage through the BBB. Post sacrificial analysis of felines at 48 hours indicate widespread distribution of enzyme activity throughout brain tissue, with βgal levels as high as 17-fold over untreated in the occipital cortex, 11-fold over untreated in the cerebellum, and 13-fold over untreated in the thalamus. When increasing to a dose of 24000 mg/kg, βgal activities reach up to 90% of normal in the cerebellum. However, spinal cord analysis indicates limited increases βgal activity compared to untreated felines.

In vitro studies indicate that only approximately 30% of βgal is released from polymersomes. In an attempt to increase effectiveness of polymersomes at lower doses, polymersomes are currently being made using hyaluronic acid (HA), a biopolymer that is a substrate for hexosaminidase A which is upregulated in GM1 gangliosidosis felines. By using both HA and PLA, we see that polymersomes are both enzymatically and pH responsive. This has increased the delivery amount of active βgal and is being translated to in vitro and in vivo studies. Furthermore, work is being done to understand if upregulated hexosaminidase A is a potential biomarker of GM1 gangliosidosis. Currently, hexosaminidase A appears to be linearly correlated with levels of autophagic failure, meaning that it could provide information on how far progressed the disease is on a patient by patient level.

Results demonstrate strict control over carrier size formed, therapeutic payload, release location, and ligand attachment possible when utilizing polymersomes as an enzyme delivery vehicle. This novel carrier provides a brain delivery platform for currently untreatable diseases and has the potential to cause a paradigm shift in the way we treat the central nervous system. Initial animal studies are highly encouraging towards the goal of creating the first clinical treatment for GM1 gangliosidosis, using a combination of ERT and nanotechnology methods to cross the BBB and deliver active enzyme.