Research ArticleBLOOD-BRAIN BARRIER

Brain delivery and activity of a lysosomal enzyme using a blood-brain barrier transport vehicle in mice

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Science Translational Medicine  27 May 2020:
Vol. 12, Issue 545, eaay1163
DOI: 10.1126/scitranslmed.aay1163

Transport vehicle for CNS therapeutics

Delivering biotherapeutics to the brain is complicated by the presence of the blood-brain barrier (BBB). Kariolis et al. and Ullman et al. developed a transport vehicle (TV) consisting of an Fc fragment engineered to bind to the transferrin receptor, a protein highly expressed at the BBB. Utilizing the TV in the form of an antibody directed against β-secretase enhanced brain delivery and effects in both mice and monkeys following systemic administration. Systemic delivery of a TV fusion with the iduronate 2-sulfatase enzyme was effective at reducing peripheral and central pathologies in a mouse model of mucopolysaccharidosis type II. The TV platform approach potentially offers a treatment for neurological disorders.

Abstract

Most lysosomal storage diseases (LSDs) involve progressive central nervous system (CNS) impairment, resulting from deficiency of a lysosomal enzyme. Treatment of neuronopathic LSDs remains a considerable challenge, as approved intravenously administered enzyme therapies are ineffective in modifying CNS disease because they do not effectively cross the blood-brain barrier (BBB). We describe a therapeutic platform for increasing the brain exposure of enzyme replacement therapies. The enzyme transport vehicle (ETV) is a lysosomal enzyme fused to an Fc domain that has been engineered to bind to the transferrin receptor, which facilitates receptor-mediated transcytosis across the BBB. We demonstrate that ETV fusions containing iduronate 2-sulfatase (ETV:IDS), the lysosomal enzyme deficient in mucopolysaccharidosis type II, exhibited high intrinsic activity and degraded accumulated substrates in both IDS-deficient cell and in vivo models. ETV substantially improved brain delivery of IDS in a preclinical model of disease, enabling enhanced cellular distribution to neurons, astrocytes, and microglia throughout the brain. Improved brain exposure for ETV:IDS translated to a reduction in accumulated substrates in these CNS cell types and peripheral tissues and resulted in a complete correction of downstream disease-relevant pathologies in the brain, including secondary accumulation of lysosomal lipids, perturbed gene expression, neuroinflammation, and neuroaxonal damage. These data highlight the therapeutic potential of the ETV platform for LSDs and provide preclinical proof of concept for TV-enabled therapeutics to treat CNS diseases more broadly.

INTRODUCTION

Lysosomal storage diseases (LSDs) comprise a diverse family of more than 50 monogenic disorders, most of which result from deficiency of a single lysosomal enzyme (1, 2). Abrogated enzyme activity leads to progressive substrate accumulation, perturbing lysosomal function and cellular homeostasis (3). Although individual LSDs are rare, it is estimated that they collectively afflict 1 in roughly 5000 newborns worldwide (4). The primary treatment for LSDs involves enzyme replacement therapies (ERTs), which have achieved variable clinical success in treating peripheral symptoms of disease. Unfortunately, more than two-thirds of LSDs are characterized by progressive central nervous system (CNS) dysfunction (5), and the inability of recombinant enzymes to cross the blood-brain barrier (BBB) renders these treatments ineffective in addressing CNS manifestations of disease (68).

Mucopolysaccharidosis type II (MPS II) is an X-linked LSD caused by deficiency of iduronate 2-sulfatase (IDS), a lysosomal enzyme involved in the catabolism of the glycosaminoglycans (GAGs) heparan and dermatan sulfate. This debilitating disease affects multiple organ systems, and the resulting clinical presentation typically includes coarse facial features, organomegaly (particularly of the liver and spleen), progressive joint and skeletal involvement, and, in the neuronopathic form of MPS II, severe cognitive deficits (9). MPS II occurs in an estimated 1 in 170,000 births (10). About two-thirds of patients display the neuronopathic phenotype, characterized by progressive cognitive impairment, behavioral symptoms, and a decreased life span, with death typically occurring within the second decade of life. The current standard of care, recombinant IDS, successfully reduces GAG accumulation in peripheral tissues but is ineffective in treating CNS components of the disease (11).

An emerging strategy to improve brain delivery of protein therapeutics has been to exploit receptor-mediated transcytosis (RMT) (12, 13). RMT is an endogenous trafficking pathway used to transport ligands such as insulin and transferrin-bound iron across the BBB through specific interactions with brain endothelial cell receptors (14, 15). As a means to improve brain exposure, protein therapeutics can be specifically engineered to bind receptors on the cerebral vasculature and use RMT to enhance their transport across the BBB and delivery into the brain parenchyma. The transferrin receptor (TfR) has long represented an attractive target to enable RMT of protein therapeutics, as this receptor is highly expressed on brain capillaries (16) and has been used to enhance CNS exposure of antibodies that bind TfR in preclinical models (1719). RMT-based delivery approaches are currently being explored to improve brain delivery of lysosomal enzymes using a variety of reported brain endothelial cell receptors, including TfR. Most of these approaches use antibody-enzyme fusions wherein both antibody Fabs bind the RMT target with an enzyme appended to the C terminus via a peptide linker (2022). These architectures result in large fusion proteins that engage RMT receptors bivalently, potentially negatively affecting brain uptake of the protein therapeutic, its subsequent CNS distribution, and native receptor trafficking (23, 24). The therapeutic potential of a standard antibody fused to a lysosomal enzyme is currently being tested in the clinic, and these approaches lead to limited reduction of substrate in cerebrospinal fluid (CSF), highlighting the need for new technologies to improve brain exposure (25, 26).

We have developed a BBB transport vehicle (TV) in which the Fc domain of the human immunoglobulin G1 (IgG1) has been engineered to bind to the TfR, creating a modular platform that may be used to enhance CNS delivery of protein therapeutics (27). Here, we demonstrate the potential of this approach to facilitate RMT of lysosomal enzymes across the BBB using an enzyme transport vehicle (ETV), in which a lysosomal enzyme has been fused to the TV. Using IDS as an initial application, we show that fusion of IDS to the TV substantially improves brain delivery of enzyme while maintaining activity and peripheral exposure. We also demonstrate that enhanced brain exposure of IDS translates to effective reduction of accumulated substrates and correction of downstream pathology in the CNS in a mouse model of MPS II disease, highlighting the therapeutic potential of the ETV approach for MPS II and potentially other neuronopathic LSDs.

RESULTS

Engineering and in vitro characterization of ETV:IDS

We recently described the BBB TV: an engineered Fc domain that binds to TfR and enhances the brain uptake and pharmacodynamic response of protein therapeutics in mouse and nonhuman primates (27). To expand the utility of the TV to treat LSDs that have CNS involvement, we developed an ETV composed of a lysosomal enzyme, IDS, fused to the TV (ETV:IDS; Fig. 1A). IDS was fused to either the N or C terminus of several different TV clones to generate a set of ETV:IDS variants with affinities to TfR around 200 to 820 nM (Fig. 1B). ETV:IDS fusions where IDS was fused to the N terminus of the TV displayed about twofold higher affinity for TfR compared to C-terminal fusions (TVe*; Fig. 1B), likely due to increased distance between the enzyme and the TfR binding site, and were prioritized for subsequent development and characterization.

Fig. 1 Engineering and in vitro characterization of ETV:IDS.

(A) ETV:IDS is a fusion of the lysosomal enzyme iduronate 2-sulfatase (IDS) to the transport vehicle, a TfR-binding Fc domain. (B) Binding affinities of ETV:IDS variants to human TfR (hTfR) were measured by steady-state surface plasmon resonance (SPR). (C) LC-MS/MS quantification of Cys84 to fGly conversion in ETV:IDS and idursulfase; n = 3. (D) Specific activities of ETV:IDS and idursulfase were measured using an in vitro fluorometric assay; n = 11 for idursulfase and n = 4 for ETV:IDS. Graphs display means ± SD (C and D). (E) 35S-labeled substrates in HEK293T IDS KO cells treated with ETV:IDS or idursulfase; n = 3. (F) 35S sulfate-labeled substrates in MPS II patient fibroblasts treated with ETV:IDS or idursulfase; n = 8 experiments with three patient lines per phenotype used in each experiment. Dashed lines in (E) and (F) correspond to amounts of GAGs or accumulated 35S-labeled substrates in wild-type or healthy control cells, respectively. (G) Binding affinities of ETV:IDS and idursulfase to M6PR were measured by ELISA; n = 2 technical replicates. Graph displays mean values ± SD. (H) M6PR- and TfR-dependent clearance of 35S-labeled substrates was assessed in MPS II patient fibroblasts treated with increasing doses of ETV:IDS or idursulfase in the presence or absence of 5 mM M6P or 3 μM TV; n = 3 experiments with three patient lines per phenotype used in each experiment. Graphs display mean values across experimental replicates ± SEM (E, F, and H). a.u., arbitrary units.

IDS has a number of posttranslational modifications that affect its biochemical and cellular activity. For example, the enzymatic activity of IDS is dependent on conversion of its active site Cys84 to formylglycine (fGly) (28). The fGly content of ETV:IDS was determined using liquid chromatography–tandem mass spectrometry (LC-MS/MS). ETV:IDS had about 50% fGly modification compared to 70% for idursulfase, a commercial IDS ERT (Fig. 1C). The resulting biochemical enzymatic activities of ETV:IDS and idursulfase were reflective of fGly content (Fig. 1D), suggesting that the difference in activity is likely due to fGly content rather than to a loss in activity from fusion to the TV.

We next generated IDS knockout (KO) human embryonic kidney (HEK) 293T cells to assess cellular activity of ETV:IDS and used an MS-based assay to quantify the individual disaccharides derived from heparan and dermatan sulfate, the GAG substrates that accumulate with IDS deficiency (fig. S1, A, C, and D) (29, 30). IDS KO cells accumulated substantially more total substrate relative to wild-type cells (fig. S1E). Treatment of IDS KO cells with ETV:IDS reduced substrates in a dose-dependent manner and lowered GAGs to amounts seen in wild-type cells (Fig. 1E). ETV:IDS had comparable potency to idursulfase in IDS KO cells [median inhibitory concentration (IC50) values of 8 and 13 pM, respectively], demonstrating that fusion of IDS to the TV did not affect its ability to degrade endogenous substrates within cells. The cellular activity of ETV:IDS was also examined in fibroblasts derived from patients with MPS II and healthy controls using a well-established 35S pulse-chase assay, in which 35S is integrated into newly synthesized GAGs (21). MPS II patient fibroblasts lack detectable IDS activity, leading to about a 10-fold accumulation of substrate and 2.5-fold accumulation of 35S signal (Fig. 1F and fig. S1, B and F). ETV:IDS was highly active in MPS II patient–derived cells, displaying a 6 pM cellular IC50 that was comparable to that of idursulfase in reducing the accumulation of 35S-labeled proteins (Fig. 1F).

The addition of mannose-6-phosphate (M6P) to N-linked glycans on IDS plays a critical role in the enzyme’s cellular uptake and trafficking, as interactions between M6P and cell surface M6P receptors (M6PRs) drive the internalization and lysosomal delivery of systemically administered IDS (22, 31). The affinity of ETV:IDS for M6PR was determined using an enzyme-linked immunosorbent assay (ELISA). ETV:IDS binds to recombinant M6PR with a median effective concentration (EC50) of 135 pM, comparable to idursulfase, which binds with an EC50 of 95 pM (Fig. 1G). Further, we demonstrated that cellular efficacy of ETV:IDS was dependent upon M6PR but not TfR, as an excess of M6P inhibited clearance of 35S-labeled proteins in treated MPS II patient fibroblasts, whereas addition of excess TV had no effect (Fig. 1H). Collectively, these data demonstrate that the M6PR-dependent trafficking and cellular activity of IDS are maintained in ETV:IDS.

Pharmacokinetics of ETV:IDS in wild-type mice

To determine whether fusion of IDS to the TV altered clearance of the enzyme, we assessed the pharmacokinetic (PK) properties of ETV:IDS and idursulfase in wild-type mice. Intravenous administration of equimolar doses revealed biphasic PK profiles for both molecules (fig. S2), where concentrations declined similarly for each molecule during the first 4 to 8 hours. Fusion of IDS to the TV, which confers neonatal Fc receptor (FcRn) binding and the potential for lower clearance due to endosomal recycling, resulted in drug clearance of 396 to 528 ml/day per kilogram for ETV:IDS compared to 1040 to 1330 ml/day per kilogram for idursulfase and a terminal half-life of 10 to 22 hours compared to 4.0 to 4.3 hours relative to idursulfase.

Reduction of GAG accumulation in peripheral tissues and fluids of IDS KO mice

We next examined whether ETV:IDS could lower peripheral GAGs in a previously described mouse model of MPS II (32, 33). Substantial GAG accumulation in tissues and fluids of IDS KO mice was observed compared to wild-type littermates, including an approximate 9-fold increase in the brain and 21-fold increase in CSF GAGs (Fig. 2A). We tested whether the accumulated GAGs in IDS KO mice could be reduced acutely after a single dose of ETV:IDS. Time-course analysis in urine and serum from IDS KO mice after ETV:IDS administration demonstrated that GAG concentrations were lowered as early as 3 days and were maximally reduced after about 7 days (P < 0.01 to 0.0001; fig. S3, A and B). On this basis, we assessed the distribution of ETV:IDS and its ability to reduce GAGs in tissues of IDS KO mice 7 days after a single dose of ETV:IDS (40 mg/kg). Distribution of ETV:IDS in the liver, spleen, and lung was confirmed at 2 hours after dosing (fig. S3C), and significant substrate reduction was observed in these tissues (P < 0.0001; Fig. 2B).

Fig. 2 ETV:IDS reduces GAG accumulation in peripheral tissues and fluids of IDS KO mice and improves brain uptake in TfRmu/hu KI mice.

(A) Total GAGs in tissues and fluids from 3-month-old IDS KO and wild-type (Wt) mice were measured by LC-MS/MS; n = 4 to 5. Data shown as means ± SEM with P values: multiple t test analysis with Holm-Sidak multiple comparison test. (B) Total GAGs in peripheral tissues from IDS KO mice were assessed 7 days after administration of a single dose of ETV:IDS (40 mg/kg) compared to vehicle-treated IDS KO and wild-type mice; n = 8 for IDS KO groups and n = 3 for wild-type groups. Data shown as means ± SEM with P values: one-way ANOVA with Dunnett’s multiple comparison test. (C) Liver and brain concentrations of ETV:IDS or Fc:IDS in TfRmu/hu KI mice after a single intravenous injection of dose (50 mg/kg); n = 4 to 5, graphs display means ± SEM with P values: multiple t tests with Holm-Sidak multiple comparison test; **P < 0.01 and ****P < 0.0001.

Distribution of ETV:IDS in brains of TfRmu/hu knock-in mice

Having demonstrated the ability of ETV:IDS to reduce peripheral GAG accumulation in a mouse model of MPS II, we next explored whether peripherally administered ETV:IDS could be effectively delivered to the brain. Because the engineered Fc domain of ETV binds to human but not mouse TfR, the wild-type and IDS KO mouse models could not be used to evaluate TfR-mediated brain uptake. To overcome this limitation, we developed a knock-in (KI) mouse model expressing a chimeric human/mouse TfR that harbors the human TfR apical domain (TfRmu/hu KI mice) that enables assessment of the brain uptake of TV-based therapeutics in murine models (27). TfRmu/hu KI mice were dosed intravenously with ETV:IDS (50 mg/kg) or Fc:IDS (50 mg/kg), a control in which a single IDS moiety was fused to a wild-type Fc domain that cannot bind TfR, to assess the impact of TfR binding on the biodistribution of ETV:IDS. Serum concentrations were similar for each molecule up to 4 hours after dosing, with some evidence of divergence after 8 hours (fig. S4). Uptake of the intact fusion protein in the liver was equivalent for both molecules (Fig. 2C), suggesting that incorporation of TfR binding does not alter liver exposure. Moreover, we observed significantly increased brain uptake of ETV:IDS relative to Fc:IDS (P < 0.01 at 2 hours and P < 0.0001 at 8 hours; Fig. 2C).

GAG reduction in the brain and peripheral tissues of IDS KO; TfRmu/hu KI mice after systemic administration of ETV:IDS

We generated an IDS-deficient mouse model that harbors the human TfR apical domain (IDS KO; TfRmu/hu KI) to test whether systemic administration of ETV:IDS effectively reduces substrate accumulation in the CNS. Western blot analysis confirmed that IDS deletion did not affect TfR expression in brain (fig. S5). Either single or four weekly activity-equivalent doses of ETV:IDS or idursulfase (747 μmol of product/min per kg or 40 mg/kg and 14.2 mg/kg, respectively) were intravenously administered to IDS KO; TfRmu/hu KI mice. After a single dose, IDS concentrations in serum were similar for ETV:IDS and idursulfase and both molecules distributed to peripheral tissues; however, we observed increased brain uptake of ETV:IDS compared to idursulfase (Fig. 3, A and B). Both ETV:IDS and idursulfase effectively lowered accumulated GAGs in the liver and spleen after 1 week of dosing, with a sustained response observed after four weekly doses (Fig. 3C, top). Treatment with idursulfase failed to reduce GAGs in brain after a single dose or repeated administration (Fig. 3C, bottom). ETV:IDS was highly effective at lowering GAGs in the CNS, eliciting a reduction of 58 and 75% in the brain and CSF, respectively, 7 days after dosing (Fig. 3C, bottom, and fig. S6). The pharmacodynamic response of ETV:IDS was sustained after repeat dosing, with a greater than 70% reduction in brain GAGs observed 1 week after the fourth dose.

Fig. 3 ETV:IDS reduces GAGs in the brain and peripheral tissues of IDS KO; TfRmu/hu KI mice.

IDS KO; TfRmu/hu KI mice were administered a single intravenous injection of ETV:IDS (40 mg/kg) or idursulfase (14.2 mg/kg; 747 μmol of product/min per kilogram of activity equivalent dose) or four weekly doses as described. The concentration of IDS in serum (A) and tissues (B) was measured after a single dose. Tissue concentration is shown 2 hours after dose; n = 4. Graphs display means ± SEM with P values: unpaired t test analysis. (C) Total GAGs in brain, CSF, and peripheral tissues of IDS KO; TfRmu/hu KI mice were measured 7 days after a single dose or multiple doses of ETV:IDS or idursulfase and compared to vehicle treatment and TfRmu/hu KI mice; n = 7 to 8 per IDS KO; TfRmu/hu KI group and n = 5 for TfRmu/hu KI group. Graphs display means ± SEM and P values: one-way ANOVA with Tukey’s multiple comparison test; *P < 0.05, **P < 0.01, and ****P < 0.0001.

ETV:IDS reduces GAGs in neurons, astrocytes, and microglia of IDS KO; TfRmu/hu KI mice

Although analysis of GAG reduction in brain lysate suggests efficacy in the CNS, it does not definitively demonstrate that ETV:IDS distributes throughout the brain parenchyma and reduces accumulated GAGs equivalently across key CNS cell types. We developed a fluorescence-activated cell sorting (FACS)–based method to isolate enriched populations of neurons, astrocytes, and microglia (Fig. 4A) to assess the distribution and efficacy of ETV:IDS in specific cell types throughout the brain. Whole mouse brains were digested, and populations of neurons, astrocytes, and microglia were subsequently enriched by FACS using a combination of positive gating on cell type–specific markers and stringent negative selection against markers of oligodendrocytes and endothelial cells (fig. S7A). To confirm the enrichment and purity of isolated cell populations, gene expression profiles for each sorted population were analyzed using RNA sequencing (RNA-seq) and compared to known profiles (Fig. 4B and fig. S7, C to D) (3438). We confirmed strong enrichment of corresponding cell type–specific genes in isolated populations and a depletion of gene markers of endothelial cells and oligodendrocytes, demonstrating that enriched populations of neurons, astrocytes, and microglia were obtained.

Fig. 4 ETV:IDS reduces GAGs in neurons, astrocytes, and microglia of IDS KO; TfRmu/hu KI mice.

(A) Schematic of FACS experiment used to isolate pure populations of neurons, astrocytes, and microglia and downstream end points analyzed. (B) Expression of cell-specific markers identified from the literature in purified neurons, astrocytes, microglia, and the input cell suspension determined by RNA-seq. Rows correspond to marker gene grouped by cell type: endothelial (endo.) and oligodendrocytes (oligo.); columns represent individual animals. Colors depict log2 fold enrichment/depletion in each sample relative to the mean expression of the gene in the unsorted (input) samples. (C) LC-MS/MS quantification of GAGs in enriched CNS cell populations; n = 3 to 5 mice. Graph displays means ± SEM normalized to fold change (FC) over TfRmu/hu KI mice; unpaired Student’s t test; *P < 0.05 and ***P < 0.001. (D) Distribution of ETV:IDS in FACS-enriched neurons, astrocytes, and microglia 2 hours after dose in vehicle-treated TfRmu/hu KI and ETV:IDS-treated IDS KO; TfRmu/hu KI mice; n = 4 mice per group. Graph displays means ± SEM; two-way ANOVA with Sidak’s test; **P < 0.01, ***P < 0.001, and ****P < 0.0001. (E) Assessment of GAGs in neurons, astrocytes, and microglia in IDS KO; TfRmu/hu KI mice after four weekly doses of ETV:IDS (40 mg/kg) or vehicle; n = 4 to 6 mice per treatment. Graph displays means ± SEM; one-way ANOVA with Tukey’s multiple comparison test; *P < 0.05 and **P < 0.01 and ****P < 0.0001. ns, not significant.

We applied this method to assess the cell type–specific distribution and efficacy of ETV:IDS in the brains of IDS KO; TfRmu/hu KI mice. GAG concentrations were substantially elevated in microglia and astrocytes isolated from IDS KO; TfRmu/hu KI mice relative to TfRmu/hu KI controls, with a modest (albeit nonsignificant) elevation observed in neurons (Fig. 4C). We dosed IDS KO; TfRmu/hu KI mice intravenously with ETV:IDS (40 mg/kg) and assessed enzyme concentration across CNS cell populations 2 hours after dosing. Treatment with idursulfase was not included for direct comparison as a lack of efficacy on a whole-brain level (Fig. 3C) obviated the need for cell type–specific analysis. Accumulation of ETV:IDS was observed in neurons, astrocytes, and microglia, confirming that ETV:IDS effectively distributed to the brain parenchyma and was taken up by key CNS cell types (Fig. 4D). We next dosed IDS KO; TfRmu/hu KI mice with ETV:IDS (40 mg/kg) intravenously once a week for 4 weeks and assessed the ability of ETV:IDS to reduce GAG accumulation across CNS cell types. In this study, with larger group sizes, significant accumulation of GAGs was observed across cell types, including neurons (P < 0.05 for neurons, P < 0.01 for astrocytes, and P < 0.0001 for microglia; Fig. 4E). ETV:IDS treatment reduced GAGs in all three cell types to concentrations comparable to that seen in TfRmu/hu KI mice after repeated administration, illustrating that the TV platform enables delivery of the enzyme past the cerebral endothelium to brain cells and that this delivery is sufficient for efficacy across key CNS cell types (Fig. 4E).

Reduction of lysosomal lipid accumulation in brains of IDS KO; TfRmu/hu KI mice

The primary accumulation of substrates observed in LSDs ultimately contributes to compromised lysosomal function, leading to the buildup of secondary lysosomal storage products (39). Elevated amounts of lysosomal proteins and lipids, including gangliosides (GMs), have been observed in the brains of IDS KO mice and patients with MPS II (40, 41). We used an LC-MS/MS–based assay to quantify a panel of lipids that commonly accumulate as a result of lysosomal dysfunction (tables S1 to S5). Several ganglioside, glucosylceramide, and bis(monoacylglycerol)phosphate (BMP) species were elevated in the brains of IDS KO; TfRmu/hu KI mice compared with TfRmu/hu KI controls (Fig. 5A and table S6). ETV:IDS effectively reduced secondary lysosomal lipid accumulation, lowering amounts of these lipids to those seen in control mice after four weekly doses of ETV:IDS (Fig. 5, A to C). As was observed for GAG accumulation in the brain, idursulfase treatment did not affect lysosomal lipids after repeated intravenous administration of an activity-equivalent dose of enzyme. Differential lysosomal lipid dysregulation was also observed on a cellular level in isolated neurons, astrocytes, and microglia (Fig. 5D and fig. S8). The extent and nature of lipid accumulation was cell type specific as, for example, astrocytes and neurons showed no increase in total BMP or gangliosides accumulation, respectively (fig. S8). Treatment with ETV:IDS normalized lysosomal lipid storage in isolated neurons, astrocytes, and microglia (Fig. 5D and fig. S8), providing additional support that ETV:IDS can correct secondary lysosomal dysfunction in the brain parenchyma.

Fig. 5 ETV:IDS reduces lysosomal lipid accumulation in the brain of IDS KO; TfRmu/hu KI mice.

IDS KO; TfRmu/hu KI mice were administered four weekly intravenous injections of ETV:IDS (40 mg/kg) or idursulfase (14.2 mg/kg; activity-equivalent dose) as described. Lipids from a targeted panel were measured in the brains of TfRmu/hu KI mice or IDS KO; TfRmu/hu KI mice treated with vehicle, ETV:IDS, or idursulfase. (A) A heatmap of a panel of lysosomal lipid species was generated by normalizing the average for each group to that observed in TfRmu/hu KI mice. White depicts wild-type amounts, red shows an accumulation, and blue shows a reduction compared to TfRmu/hu KI mice. Representative plots are shown for gangliosides (B), glucosylceramides (C; left), and BMP (C; right) in the brains of IDS KO; TfRmu/hu KI mice after multiple doses of ETV:IDS or idursulfase, compared to vehicle treatment and TfRmu/hu KI mice; n = 8 per IDS KO; TfRmu/hu KI group and n = 5 for the TfRmu/hu KI group. (D) Amounts of GM3 (d36:1) are shown from isolated neurons, astrocytes, and microglia from IDS KO; TfRmu/hu KI mice after multiple doses of ETV:IDS, compared to vehicle treatment and TfRmu/hu KI mice; n = 6 per IDS KO; TfRmu/hu KI group and n = 4 for the TfRmu/hu KI group. (E) Matrix-assisted laser desorption/ionization MS images were acquired from coronal brain sections of TfRmu/hu KI and IDS KO; TfRmu/hu KI mice after four weekly doses of vehicle, idursulfase, or ETV:IDS. Representative images were obtained for select ganglioside species. The top panel shows the distribution of the signal at a mass/charge ratio (m/z) of 1382.816, corresponding to GM2 (d36:1), whereas the bottom panel shows the distribution of the signal at a m/z of 1179.738, corresponding to GM3 (d36:1). Images depict the relative intensity of each signal from 0 to 100%. Graphs display means ± SEM and P values: one-way ANOVA with Dunnett’s multiple comparison test; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

We next performed MS imaging (MSI) to determine the spatial distribution of lipid accumulation in brains of IDS KO; TfRmu/hu KI mice and to assess whether ETV:IDS administration could correct lysosomal lipid accumulation throughout the brain. We observed an enrichment of several species of gangliosides in the brains of IDS KO; TfRmu/hu KI mice compared with TfRmu/hu KI controls, similar to results observed in our analysis of homogenized tissues (Fig. 5E). Ganglioside accumulation was region specific and concentrated in the hypothalamus and amygdala brain regions. Four weekly doses of ETV:IDS qualitatively reduced the accumulation of these ganglioside species throughout all brain regions assessed to amounts seen in TfRmu/hu KI mice, whereas only modest reductions in gangliosides were observed with idursulfase treatment (Fig. 5E).

Reduction of microglial activation in IDS KO; TfRmu/hu KI mice

Neuroinflammation represents a common hallmark of many neuronopathic LSDs (42) and has been reported in mouse models of MPS II disease and in patients with MPS (4345). We quantified two markers of microglia activity, CD68 and triggering receptor expressed on myeloid cells 2 (Trem2), through immunohistochemical analysis of brain tissue sections or biochemical analysis of brain lysates, respectively. CD68 and Trem2 expression were both elevated in the brains of IDS KO; TfRmu/hu KI mice compared to TfRmu/hu KI controls (P < 0.001 to 0.0001; Fig. 6, A to C). Four weekly doses of ETV:IDS (40 mg/kg) effectively reduced the CD68 signal throughout the brains of IDS KO; TfRmu/hu KI mice (Fig. 6, A to B). In addition, Trem2 in the brain was reduced to amounts seen in TfRmu/hu KI mice after repeated systemic administration of ETV:IDS (Fig. 6C). Four weekly activity-equivalent doses of idursulfase, however, failed to normalize CD68 and Trem2 expression (Fig. 6, A to C).

Fig. 6 ETV:IDS reduces markers of microglial phenotype in the brain of IDS KO; TfRmu/hu KI mice.

IDS KO; TfRmu/hu KI mice were administered four weekly intravenous injections of ETV:IDS (40 mg/kg) or idursulfase (14.2 mg/kg; 747 μmol of product/min per kilogram of activity equivalent dose) as described. (A) Immunofluorescence staining was performed for 4′,6-diamidino-2-phenylindole (DAPI; blue) and CD68 (green), a marker of activated macrophages and microglia, from the brains of TfRmu/hu KI and IDS KO; TfRmu/hu KI mice treated with vehicle, ETV:IDS, or idursulfase. Representative images (magnification, ×20) were obtained from the indicated brain regions: hippocampus (top), cortex (middle), and thalamus (bottom). (B) The total area of CD68 staining within defined regions within the hippocampus, cortex, and thalamus was quantified and normalized to the total area of tissue analyzed. (C) Amounts of Trem2, a marker of microglial activity, were measured in the brains of IDS KO; TfRmu/hu KI mice after ETV:IDS or idursulfase treatment and compared to vehicle treatment and TfRmu/hu KI mice; n = 8 per IDS KO; TfRmu/hu KI group and n = 5 for the TfRmu/hu KI group. Graphs display means ± SEM and P values: one-way ANOVA with Dunnett’s multiple comparison test; ***P < 0.001 and ****P < 0.0001.

Correction of perturbed gene expression in IDS KO; TfRmu/hu KI mice

We demonstrated that IDS deficiency leads to substantial alterations in gene expression as assessed at the whole brain level, which are largely driven by perturbations specifically in microglia (Fig. 7A). RNA-seq analysis of gene expression in microglia from IDS KO; TfRmu/hu KI mice revealed increased expression of disease-associated microglia (DAM) genes compared to cells from TfRmu/hu KI mice (gene set enrichment, P < 5 × 10−61; Fig. 7B). Alterations in the expression of DAM genes are consistent with changes observed in reactive microglia from mouse models of adult neurodegenerative disease, including Alzheimer’s disease and amyotrophic lateral sclerosis (ALS) (46), suggesting a neurodegenerative phenotype in MPS II mice. ETV:IDS restores gene expression in IDS-deficient mice, attenuating the up-regulation of DAM gene expression in microglia after 4 weeks of dosing (gene set enrichment, P < 4 × 10−69) and leading to a microglial gene expression profile similar to that observed in TfRmu/hu KI mice (Fig. 7, A and B). In addition, expression of genes associated with lysosomal homeostasis [coordinated lysosomal expression and regulation (CLEAR) genes] (47, 48) was increased in microglia from IDS KO; TfRmu/hu KI mice compared to TfRmu/hu KI mice (gene set enrichment, P < 4 × 10−28; Fig. 7C) and similarly attenuated with ETV:IDS treatment (gene set enrichment, P < 2 × 10−33; Fig. 7C).

Fig. 7 ETV:IDS corrects perturbations in gene expression in microglia isolated from IDS KO; TfRmu/hu KI mice.

(A) Volcano plots for cell input for FACS analysis (left) and microglia (right) demonstrate differential gene expression between the TfRmu/hu KI and IDS KO; TfRmu/hu KI mice that is corrected upon treatment with ETV:IDS. Each point corresponds to a single gene, and those highlighted in red were up- or down-regulated in the absence of ETV:IDS with a false discovery rate (FDR) of <10% and an absolute log2 fold change of >1 (dashed lines), n = 4 to 6 mice per group. Expression of (B) DAM signature and (C) CLEAR network genes in FACS-isolated microglia. Rows correspond to genes; columns represent individual animals. Colors depict log2 fold enrichment/depletion in each sample relative to the mean expression of the gene in the TfRmu/hu KI group.

Prevention of neuroaxonal injury in IDS KO; TfRmu/hu KI mice after chronic dosing with ETV:IDS

Secondary lysosomal dysfunction and perturbed glial activation are believed to ultimately contribute to the progressive degeneration of neurons observed across neuronopathic LSDs (5, 49). Neurofilament light chain (NfL), a specific subunit of the neuronal cytoskeleton that is released into CSF after axonal damage, has emerged as a sensitive marker of neurodegeneration (50, 51). In support of this, elevated amounts of NfL in CSF have been observed in patients across several adult neurodegenerative diseases, including Alzheimer’s disease and frontotemporal dementia (52, 53). IDS KO; TfRmu/hu KI mice were administered 13 weekly doses of ETV:IDS to determine whether systemic administration of ETV:IDS could prevent neuronal injury over time using NfL amounts as a readout of axonal damage. Dosing of ETV:IDS in the work reported here ranged from single and four weekly doses of 10 and 40 mg/kg for initial proof-of-concept studies (Figs. 3 to 7). We performed a further dose-ranging study where IDS KO; TfRmu/hu KI mice were administered ETV:IDS (1, 3, or 10 mg/kg), with quantification of brain exposure at 2 hours after dose; the results demonstrated dose-dependent brain exposure across the entire range (2.5 nM at 1 mg/kg dose; 5 nM at 3 mg/kg; and 8.5 nM at 10 mg/kg; fig. S9). Given the chronic dosing paradigm used to assess neuronal damage, ETV:IDS (1 or 3 mg/kg) was therefore used to determine the extent of GAG and lysosomal lipid reduction achieved with clinically relevant doses of enzyme. Thirteen weekly doses of ETV:IDS significantly reduced GAG accumulation in the liver, brain, and CSF (P < 0.01 to 0.0001; Fig. 8, A to C), and further, completely corrected glucosylceramide, BMP, and GM3 accumulation even at the lowest dose tested (Fig. 8D and fig. S10). Increased amounts of NfL were observed in CSF from IDS KO; TfRmu/hu KI mice, and reduction was achieved after prolonged treatment with ETV:IDS (Fig. 8E). These data demonstrate that ETV:IDS can effectively attenuate neuroaxonal injury in a mouse model of disease and support its potential to correct disease-relevant dysfunction downstream of GAG accumulation.

Fig. 8 ETV:IDS reduces GAGs, lysosomal lipids, and neuroaxonal injury upon chronic dosing.

IDS KO; TfRmu/hu KI mice were administered 13 weekly intravenous injections of ETV:IDS (1 or 3 mg/kg) as described. (A to C) LC-MS/MS quantification of GAGs in the liver (A), brain (B), and CSF (C) was measured after 13 weekly doses of ETV:IDS and compared to vehicle treatment and TfRmu/hu KI mice. (D) Glucosylceramide, BMP, and GM3 ganglioside amounts were measured in brains of IDS KO; TfRmu/hu KI and TfRmu/hu KI mice after chronic dosing. (E) Neurofilament light chain (NfL) in CSF of mice was measured from IDS KO; TfRmu/hu KI and TfRmu/hu KI mice after chronic dosing; n = 9 to 10 per IDS KO; TfRmu/hu KI groups and n = 10 for the TfRmu/hu KI group. One outlier was removed from TfRmu/hu vehicle-treated and DNL310 (3 mg/kg)–treated groups as determined by Grubbs test or the ROUT method. Graphs display means ± SEM and p values: one-way ANOVA with Dunnett’s multiple comparison test; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

DISCUSSION

Although most LSDs display progressive CNS dysfunction, there remains a paucity of treatments that effectively address the neurological manifestations of these diseases. Currently, a single approved therapy exists to treat a neuronopathic LSD by intracerebroventricular administration, and a clinical trial using intrathecal administration of IDS for treatment of MPS II is underway (54, 55). However, these direct CNS delivery approaches are invasive, susceptible to device issues, and have the potential for suboptimal brain distribution (54). Additional therapeutic strategies under exploration to increase CNS exposure of lysosomal enzymes include hematopoietic stem cell transplantation, gene therapy, and biotherapeutics engineered to increase the brain exposure of enzymes via RMT (44, 56, 57). Here, we describe the design and preclinical proof of concept of ETV:IDS, composed of the lysosomal enzyme IDS fused to a BBB TV, for the treatment of MPS II disease.

ETV:IDS was engineered to bind to TfR, a receptor highly expressed on brain microvessel cells, in a monovalent manner, with an affinity optimized for maximal brain uptake. This is in contrast to alternative anti-TfR binding antibody-enzyme fusions that engage multiple copies of TfR with an apparent affinity in the picomolar range (22). Clustering of TfR has been shown to alter receptor trafficking, leading to TfR degradation and iron dysregulation in vitro (58, 59). Furthermore, high-affinity binding decreases the efficiency of TfR-mediated brain transport and similarly drives receptor degradation in vivo (60). We establish that ETV:IDS, by the manner in which it has been engineered to interact with TfR, improves delivery of IDS to the brain without affecting the native properties of the enzyme necessary for efficient lysosomal targeting and function or the native trafficking of TfR. Collectively, this translates to reductions of accumulated substrates in both brain and peripheral tissues and supports the potential of ETV:IDS as an intravenous monotherapy to treat the peripheral and CNS manifestations of MPS II.

In addition to trafficking across brain endothelial cells, effective distribution of enzyme throughout the brain parenchyma and uptake into neurons, microglia, and astrocytes will likely be required to modify CNS disease progression in neuronopathic LSDs. Current approaches assessing brain exposure of protein therapeutics largely rely on whole-brain analysis of drug concentration that fails to differentiate between distribution in endothelial cells and brain parenchymal cells (22, 61). To address this concern, we used a FACS-based isolation method to demonstrate that accumulation of GAGs occurs in neurons, astrocytes, and microglia in a mouse model of MPS II, although to varying degrees. Collectively, these data suggest that dysfunction across cell types may play a role in the CNS manifestations of MPS II. We showed that ETV:IDS was delivered to neurons, astrocytes, and microglia and completely corrected the observed GAG accumulation in these cells, illustrating that fusion of IDS to the TV enables distribution to the brain parenchyma and, ultimately, efficacy in major CNS cell types. Because the regional and cell type–specific distribution of lysosomal enzymes throughout the brain is dictated both by route of administration and interactions with cell-surface receptors, additional studies are warranted to compare the distribution and efficacy achieved with the ETV platform to that observed with intrathecally administered ERTs and other RMT-based approaches targeting other brain endothelial cell receptors and/or having different receptor binding affinities.

Deficiency of an individual lysosomal enzyme often leads to storage of undegraded macromolecules in addition to accumulation of the enzyme’s primary substrates, and these secondary storage products themselves can further contribute to perturbed endolysosomal function. For example, accumulation of lysosomal lipids in the brain is a common hallmark observed across a number of neuronopathic LSDs irrespective of the primary deficiency (3). Secondary lysosomal storage products beyond elevated GAGs, including glycosphingolipids and BMP, have been reported in mouse models of MPS II and in postmortem brain tissue from patients with MPS II (40, 41, 44, 62, 63). Through targeted LC-MS/MS–based analysis of lysosomal lipids, we identified a panel of lysosomal lipid species that accumulate in brains of IDS-deficient mice that included the gangliosides GM2 and GM3 and several species of glucosylceramides and BMP. Repeated administration of ETV:IDS reduced accumulation of these lysosomal lipids to amounts seen in control mice, suggesting that the observed brain GAG reduction translates to full correction of secondary lysosomal storage. Reduction in lysosomal lipids was demonstrated in brain parenchymal cells and suggests that ETV:IDS can correct lysosomal dysfunction broadly across key cell types throughout the brain. MSI analysis of gangliosides further supports this conclusion by demonstrating that the concentrated accumulation of GM2 and GM3 species in the hypothalamus and amygdala of IDS-deficient mice was completely corrected upon ETV:IDS treatment. Perhaps expectedly, repeated administration of idursulfase led to a mild reduction of GM2 and GM3 species in the ventral hypothalamus. The ventromedial arcuate and median eminence within this region have been previously shown to be a circumventricular area with highly permeable, fenestrated vasculature, suggesting that systemic administration of enzyme alone may have some limited activity in specific CNS sites that lack a BBB (6467). Fusion of IDS to the TV was required for widespread correction of ganglioside accumulation throughout the brain.

Excessive accumulation of primary substrates and secondary storage products can trigger a pathogenic cascade within the brain, leading to increased glial activation and degeneration of neurons. Activated microglia are a common pathological feature of several neurodegenerative diseases in addition to neuronopathic LSDs, and perturbed housekeeping and sensing functions of microglia are thought to ultimately contribute to neuronal cell death (68). Lysosomal function is particularly critical for all phagocytes, including microglia, as phagocytic activity must be matched with robust lysosomal degradative capacity so as to enable efficient catabolism of phagocytic cargoes. Hence, LSDs are expected to profoundly affect microglial function, and accordingly, recent studies in mouse models of disease suggest that microglial inflammatory responses play a role in the pathogenesis of several MPS diseases and widespread microgliosis, up-regulated expression of proinflammatory genes, and an increase in inflammatory cytokines have been observed in IDS-deficient mice (44, 45, 56, 69). Elevated proinflammatory cytokines have also been reported in plasma and CSF from patients with MPS (7072). We observed substrate accumulation and increased expression of CD68 and Trem2, two markers of microglia activity, in the brains of IDS-deficient mice and achieved complete normalization of both markers after repeated administration of ETV:IDS. Trem2 is a cell-surface receptor that can be cleaved to produce a soluble protein fragment (sTrem2), and elevated amounts of sTrem2 in CSF have been observed in neurodegenerative diseases, including Alzheimer’s disease (73). Beyond alterations in CD68 and Trem2, gene expression in microglia from IDS-deficient mice was substantially perturbed, including altered expression of genes associated with neurodegenerative disease (DAM genes) and lysosomal homeostasis (CLEAR network genes). All of these changes were attenuated upon ETV:IDS treatment. These data are consistent with previously published whole-brain RNA-seq analysis, which suggested that inflammatory pathways are perturbed in IDS KO mice and highlight the potential contributions of microglial dysfunction in MPS II disease (74). Additional information is needed to understand the extent of inflammation observed in MPS II patient brains.

There is an emerging consensus that glial activation, commonly reported in mouse models of MPS II disease and in patients with MPS, may contribute to progressive degeneration of neurons throughout the brain in MPS disorders (4345). Elevation of NfL, a marker of axonal degeneration, was observed in the CSF from IDS KO; TfRmu/hu KI mice and has been reported in various neurodegenerative diseases (50, 53). Increased NfL in CSF showed a dose-dependent reduction after chronic dosing, suggesting that ETV:IDS can prevent axonal degeneration. These data demonstrate that ETV:IDS can effectively attenuate neurodegeneration in a mouse model of disease and support the potential of ETV:IDS to correct disease-relevant dysfunction downstream of GAG accumulation. Future studies are warranted to assess whether NfL is elevated in CSF from patients with MPS II and if NfL may serve as a potential biomarker of neuroaxonal injury across LSDs more broadly.

Our study is not without its limitations. Although the FACS-based method used allows the analysis of enzyme delivery and correction of disease-relevant dysfunction across neurons, astrocytes, and microglia, this method relies on specific cell-surface markers to enable the isolation of enriched CNS cell populations. Hence, we cannot rule out the possibility that the reported improvement of GAG and lipid storage and perturbed gene expression reflects correction in specific subpopulations of each of these cell types. Additional studies that use diverse cell type–specific surface markers are warranted to more completely understand the effects of ETV:IDS on neurons, astrocytes, and microglia across brain regions and cell subtypes. Further, although the mouse models used in this work recapitulate many aspects of MPS II, there are obvious limitations associated with correlating end points in these models to those used to evaluate clinical efficacy. Similarly, many of the downstream biomarkers and end points used to assess efficacy in this study remain exploratory and have not yet been clinically validated. Ultimate proof of concept for ETV:IDS awaits evaluation in patients with MPS II.

In conclusion, we demonstrate that systemic administration of ETV:IDS can reduce accumulated primary substrates and correct downstream pathologies in the brain of a mouse model of MPS II disease. Given the strong preclinical proof-of-concept data supporting its therapeutic potential, ETV:IDS is currently in clinical development for the treatment of neuronopathic MPS II disease (clinical trial ID, NCT04251026). Together with the inherent modularity of the platform, these data also suggest that the ETV can be harnessed more broadly to enhance brain delivery of additional lysosomal enzymes and proteins, with the potential to yield a new class of promising biotherapeutics for the treatment of CNS disease.

MATERIALS AND METHODS

Study design

This study was designed to engineer and characterize ETV:IDS—a fusion between the lysosomal enzyme IDS and our BBB TV, an Fc domain engineered to interact with the TfR. We first engineered the ETV by generating various fusions between the TV and IDS and characterizing the resulting molecules’ biophysical and biochemical properties. We then evaluated the in vivo biodistribution of ETV:IDS compared to idursulfase in our KI mouse model expressing a chimeric human/mouse TfR that harbors the human TfR apical domain. Lastly, we studied the efficacy of ETV:IDS on disease-relevant end points, both proximal by quantifying substrates (GAGs) and distal by measuring effects on lysosomal lipids, microglial activation, and neuronal injury in our IDS-deficient mouse models.

Sample sizes for in vivo studies were determined on the basis of statistical power calculations from prior studies and past experiences with the models and end points used. Figure legends contain sample sizes, replicate information, and statistical tests used. Outliers were calculated by applying the Grubbs test or robust regression and outlier removal (ROUT) method and are reported in data file S1. For all in vivo studies, animals were randomly assigned to groups before experiments, and researchers were blinded to treatment when possible during quantification and analysis.

Statistical analysis

Data have either been expressed as means ± SEM or SD as indicated in figure legends. All statistical analysis was performed in GraphPad Prism 8. Analysis was performed using multiple t test, with Holm-Sidak multiple comparison test, one-way analysis of variance (ANOVA) with Dunnett multiple comparison, one-way ANOVA with Tukey’s multiple comparison test, unpaired t test, or two-way ANOVA with Sidak’s test, as indicated in figure legends. Criterion for differences to be considered significant was P < 0.05. Gene expression analysis was performed using the limma empirical Bayes analysis pipeline; gene set enrichment analysis was performed with the camera algorithm included in the limma R package.

SUPPLEMENTARY MATERIALS

stm.sciencemag.org/cgi/content/full/12/545/eaay1163/DC1

Materials and Methods

Fig. S1. Validation of LC-MS/MS methods and cellular experimental models.

Fig. S2. PK of ETV:IDS in wild-type mice.

Fig. S3. GAG reduction in fluids and peripheral tissue biodistribution of ETV:IDS in IDS KO mice.

Fig. S4. PK of ETV:IDS in TfRmu/hu mice.

Fig. S5. IDS deletion does not affect TfR levels in the brain.

Fig. S6. Individual disaccharide analysis from the brain and CSF of dosed IDS KO; TfRmu/hu KI mice.

Fig. S7. Characterization of FACS-isolated CNS cell types.

Fig. S8. Analysis of ganglioside, BMP, and glucosylceramide levels in FACS-isolated CNS cell types after treatment with ETV:IDS.

Fig. S9. Relationship between dose level and brain concentration of ETV:IDS.

Fig. S10. Analysis of lysosomal lipids in CSF after chronic treatment with ETV:IDS.

Table S1. LC-MS acquisition parameters information for the BMP and gangliosides assay.

Table S2. LC-MS acquisition parameters information for GlcCer and GalCer assay.

Table S3. LC-MS acquisition parameters information for eicosanoid assay.

Table S4. LC-MS acquisition parameters for lipidomics assay in negative ionization mode.

Table S5. LC-MS acquisition parameters for lipidomics assay in positive ionization mode.

Table S6. Targeted lipidomic analysis of IDS KO; TfRmu/hu KI mice after repeated administration of ETV:IDS or idursulfase.

Data file S1. Raw data.

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REFERENCES AND NOTES

Acknowledgments: We thank Y. Paila, D. Demian, and J. Goldovitz (Lake Pharma, Hopkinton, MA) for help with the 35S protein accumulation assay and K. Lin for help in heatmap generation of lysosomal lipid alterations. We also thank J. Muenzer, S. Jones, and P. Harmatz for useful feedback on in vivo study designs and J. Lewcock for helpful feedback on the manuscript. Funding: This study was funded by Denali Therapeutics. Author contributions: Conceptualization: M.S.D., A.P.S., R.J.W., Z.K.S., M.S.K., and A.G.H. Methodology: J.C.U., A.A., J.A.G., A.B., and J.W. Formal analysis: J.A.G., S.L., V.M.D., J.C.D., T.S., and K.R.H. Investigation, J.C.U., A.A., J.A.G., A.B., C.S.M., J.W., T.G., C.B., D.J.K., J.R.B., N.L., R.R., A.A.N., S.S.D., C.H., J.D., H.L.T., R.C.W., W.K., H.S., H.N., T.E., M.D.T., J.L.H., M.L.R., A.S., G.A., and M.S.K. Writing—original draft: J.C.U., A.A., J.A.G., A.B., A.P.S., M.S.K., and A.G.H. Writing—review and editing: R.G.T., A.P.S., M.S.K., and A.G.H. Supervision: R.M.C., S.H., S.P., P.E.S., K.G., K.R.H., R.G.T., G.D.P., D.D., A.P.S., Z.K.S., M.S.K., and A.G.H. Competing interests: J.C.U., A.A., J.A.G., A.B., C.S.M., J.W., T.G., C.B., D.J.K., N.L., R.R., A.A.N., S.S.D., C.H., J.D., H.L.T., R.C.W., S.L., V.M.D., W.K., H.S., H.N., T.E., J.C.D., S.P., P.E.S., M.S.D., K.G., A.S., T.S., K.R.H., R.G.T., G.D.P., G.A., D.D., A.P.S., R.J.W., Z.K.S., M.S.K., and A.G.H. are paid employees of Denali Therapeutics Inc. Denali has filed patent applications related to the subject matter of this paper; WO 2019/070577, FUSION PROTEINS COMPRISING ENZYME REPLACEMENT THERAPY ENZYMES. R.G.T. currently serves on scientific advisory boards for Alcyone Lifesciences Inc. and a Lundbeck Foundation supported Research Initiative on Brain Barriers and Drug Delivery. Data and materials availability: All data associated with this study are present in the paper or the Supplementary Materials. The RNA-seq data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO series accession number GSE139002 (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE139002).

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