Targeting neuronal lysosomal dysfunction caused by β-glucocerebrosidase deficiency with an enzyme-based Brain Shuttle construct

Abstract

Biallelic mutations in GBA1 that lead to reduced β-glucocerebrosidase (GCase) activity result in the monogenic lysosomal storage disease Gaucher disease (GD). Variants in one GBA1 allele are the most common genetic risk factor for multiple synucleinopathies including Parkinson’s disease (PD). Therapies to increase GCase activity in the brain hold great promise for the treatment of these diseases. To this end, we have developed blood-brain barrier penetrant therapeutic molecules by fusing antibody moieties that bind the transferrin receptor (TfR) to murine or human GCase (referred to as mGCase-mBS or hGCase-hBS, respectively). We demonstrate that these fusion proteins maintain full enzymatic activity and, while their total cellular uptake is only marginally increased compared to the enzyme alone, they have up to 100-fold better lysosomal uptake and function. Uptake and efficacy of GCase-BS relies primarily on binding to the TfR, rather than to mannose phosphate receptors (M6PRs) as conventional enzyme replacement therapy. In a GD cellular model, GCase-BS rapidly rescues the lysosomal proteome and lipid accumulations beyond known GCase substrates. Intravenous injection of mGCase-mBS leads to significant reduction of brain lysosomal membrane lipids in a GD mouse model which is sustained for four weeks. Monthly dosing over six months shows sustained efficacy and reduces neurofilament-light chain (NFL) plasma levels. Collectively, these findings demonstrate the great potential of TfR-targeted GCase for treating GBA1-associated neurodegeneration, provide insight into candidate biomarkers of GD lysosomal dysfunction, and ultimately may open a new treatment paradigm for lysosomal storage diseases (LSDs) extending beyond the central nervous system (CNS).

A Fixable Fluorescence‐Quenched Substrate for Quantitation of Lysosomal Glucocerebrosidase Activity in Both Live and Fixed Cells

“…Delivery of recombi-nant enzyme to GBA1 KO cells by addition to the media, led to a transient increase in lysosomal GCase activity (Figure 3D, E) at 24 h, which diminishes over time as the enzyme is secreted from the cells into the media as the enzyme is unstable in neutral media. [47] We next showed that pharmacological perturbations to trafficking of GCase from the ER to lysosomes, induced by the known small molecules brefeldin A and monensin, [48,49] lead to a decrease in lysosomal GCase activity as measured using LysoFix-GBA (Figure S3). Further, using the pharmacological agent bafilomycin A, which leads to deacidification of lysosomes, results in an expected decrease in lysosomal GCase activity (Figure S3).…”

The SATB1‐MIR22‐GBA axis mediates glucocerebroside accumulation inducing a cellular senescence‐like phenotype in dopaminergic neurons

“…The senescence inducer S100A9, which is upregulated with age (Swindell et al., 2013), also co‐localizes and co‐aggregates with α‐SYN in 20% of Lewy bodies and 77% of neuronal cells in the substantia nigra of PD patients (Horvath et al., 2018). Additionally, GBA‐KO cells that exhibit elevated GluCer levels demonstrate upregulation of S100A9 (Gehrlein et al., 2023). This is noteworthy since S100A9 is implicated in the induction of cellular senescence (Shi et al., 2019) and is significantly upregulated in multiple aging murine and human tissues, including those in the central nervous system (Swindell et al., 2013).…”

Development of quantitative high-throughput screening assays to identify, validate, and optimize small-molecule stabilizers of misfolded β-glucocerebrosidase with therapeutic potential for Gaucher disease and Parkinson’s disease

“…For initial analysis of glucosylsphingosine (GluSph) levels in frozen pellets of untreated H4 cells, cells were lysed by addition of distilled water followed by protein precipitation with methanol, as described [8,62]. The dry lipid samples were reconstituted in a mixture of acetonitrile/water 90/10 (v/v) containing 1% DMSO.…”

“…Analysis was performed via positive ion electrospray LC-MS/MS in multiple reaction-monitoring (MRM) mode, using deuterated compounds as internal standards. A Waters Xevo-TQ-S mass spectrometer connected to a complete Waters Acquity Iclass UPLC system was used as previously described, with a mixture of acetonitrile/methanol/water 40/40/20 (v/v/v) as the wash solvent [8,62]. Samples were analyzed on a BEH glycan amide column (100 × 2.1 mm, 1.7 μm particle size; Waters Corporation, Switzerland) with a flow rate of 0.25 mL/min and an oven temperature of 30°C.…”

“…The current standard-of-care for GD is enzyme replacement therapy (ERT), whereby recombinant, mannose-terminated GCase is delivered to lipid-laden macrophages via biweekly intravenous infusions. While ERT offers dramatic reversal of the peripheral symptoms of the disease, such as hepatosplenomegaly and hematologic abnormalities [7], it is rapidly cleared from the blood and, unfortunately, does not penetrate the blood-brain barrier (BBB) [8]. Therefore, it has no effect on the neurologic manifestations seen in type 2 (acute neuronopathic) or type 3 (subacute neuronopathic) GD [9], and it does not prevent or modify the progression of parkinsonism in patients with GD [10].…”