Nanoparticle Intravenous Distributed Enzyme Replacement Therapy (NanoDIVERT) for Non-Invasive Treatment of Neurologic GM1 Gangliosidosis

Introduction: Lysosomal Storage Diseases (LSD) are caused by dysfunctional enzymes which are then unable to cleave substrates, resulting in detrimental substrate accumulation and grave prognoses. Enzyme Replacement Therapy (ERT), which provides patients with healthy enzymes, is standard practice for treatment of non-neuropathic LSDs. One LSD where this treatment is not yet an option is GM1 gangliosidosis (GM1) as it presents heavily in neuronal cells and the blood-brain barrier (BBB) prevents up to 98% of small molecules from reaching the brain, including endogenous enzymes. In GM1-afflicted patients, the enzyme ß-galactosidase (ß-gal) is either misfolded or not produced and can’t cleave the GM1 ganglioside, causing a build-up of glycosphingolipid in the lysosome. Our laboratory has utilized PEG-b-PLA polymersomes to encapsulate ß-gal to implement BBB-bypassing ERT for GM1. Tagging with Apolipoprotein E (ApoE) provides a targeting effect through the BBB by recognition of Low Density Lipoprotein Receptors (LDLR), which are upregulated in the brain during inflammation. Previous efforts from our laboratory have utilized felines with naturally occurring GM1 to observe the effects of intravenous injection of ß-gal-loaded polymersomes. These efforts produced up to a 10-fold increase in the enzyme activity of treated felines compared to untreated felines (Figure 1B). The recent development of a GM1 mouse model encourages establishment of pharmacokinetic and pharmacodynamic profiles of the NanoDIVERT system, strengthening the statistical significance of this treatment method which is relevant given the rarity of the disease. Using ß-gal-/- mice, we are examining the biodistribution and therapeutic efficacy of intravenous injection of ß-gal loaded PEG-b-PLA polymersomes.

Materials and

Methods: PEG-b-PLA polymersomes have been previously explored in our lab with great success for application in the feline model of GM1 gangliosidosis. To develop NanoDIVERT, PEG-b-PLA polymer was dissolved in dimethyl sulfoxide and then injected into a 2% mannitol in water solution using a 20-gauge needle. ApoE was dissolved in the aqueous phase for PS tagging. Diameter and surface charge changes, as determined by dynamic light scattering (DLS), were used to confirm successful ApoE tagging. Encapsulation efficiency of various fluorescently tagged proteins and dyes was examined via dialysis. Cyanine 5 (Cy5) loaded polymersomes were prepared by the same method for use with the murine model. Heterozygous ß-gal knockout mice were bred to produce ß-gal-/- mice. Mice were genotyped using ear snips and a tissue digestion assay and a touchdown polymerase chain reaction (PCR). These mice underwent pharmacokinetic studies wherein mice were injected with Cy5-loaded polymersomes at a mass of 2400mg/kg mouse body weight. Post-injection, mice were imaged using In Vitro Imaging System (IVIS) and plasma was collected via blood draws at times of 0.25, 1, 4, and 24 hours for UV-vis analysis. At the 24-hour mark, mice were sacrificed and immunofluorescence, anti-NeuN staining, and IVIS of major internal organs was performed.

Results, Conclusions, and Discussions: The polydispersity index (PDI) for untagged polymersomes and ApoE tagged polymersomes indicated polymersomes were approximately monodispersed. Polymersome diameter increased while surface charge became less negative following ApoE tagging (Figure 1A). The negative charge of the polymersomes in both cases is consistent with the expected surface charge of PEGylated structures. Cy5 dye was encapsulated at 1.7E-5 mg/mg of polymersome for in vivo analysis of pharmacokinetics. Fluorescently tagged ß-gal protein was encapsulated at 0.01 mg ß-gal/ mg polymersome for therapeutic delivery of enzyme in vivo.
Polymerase Chain Reaction on breeds of mice from two heterozygous parents produced approximately 25% mice homozygous mutants (Figure 1C). These mutants were injected with both ApoE-tagged and untagged PEG-b-PLA polymersomes encapsulated with Cy5 to determine their biodistribution and pharmacokinetic profiles. ApoE tagging enabled detectible Cy5 fluorescence in the brain after polymersome injection via tail vein, while untagged particles presented with limited neural Cy5 fluorescence. ApoE also led to higher circulation half-lives than untagged PEG-b-PLA, indicating that polymersomes spent more time in tissues before being removed, increasing their bioavailability. Previous studies on 4-week-old GM1 felines treated by our NanoDIVERT approach resulted in an increase in enzyme activity throughout the central nervous system when compared to untreated felines, bringing ß-gal enzyme activities from 10% normal to ~85% normal in the cerebellum (Figure 1). We expect to see similar results in ß-gal-/- mice.
PEG-PLA polymersomes present a unique delivery solution for enzymes in the treatment of LSDs like GM1 gangliosidosis. Tagging of polymersomes with ApoE allows targeting to LDLRs which have a higher density in brain tissue due to inflammation in GM1. ApoE enables delivery of Cy5 dye to the brain in ß-gal-/- mice, indicating its ability to enable BBB bypassing. NanoDIVERT led to increased ß-gal activity throughout the brain but most notably in the frontal and cerebral cortexes of the brain. Utilizing the GM1-affected mouse model, statistical significance can be established when mice are injected intravenously dye-loaded polymersomes for pharmacokinetics and enzyme-loaded polymersomes for pharmacodynamics. This system has the potential to change the way we treat neuropathic LSDs, beyond just GM1, by enabling non-invasive ERT to treat the brain.

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