Biomarkers for lysosomal storage diseases and methods of use thereof

Abstract

The present invention provides a method of detecting one or more biomarkers in a subject suffering from lysosomal storage disease, the method comprising: 1) measuring the concentration of a combination of two or more lipids in a sample from the subject wherein the combination of lipids is selected from the group consisting of: a) bis (monoacylglycerol) phosphate (BMP); b) a GM2 ganglioside and / or a GM3 ganglioside; c) a GD3 ganglioside; d) GD1a / b ganglioside, and d) GD1a / b ganglioside; and e) a glucosyl ceramide (GlcCer); 2) measuring the concentration of GlcCer in a sample from the subject; 3) measuring the concentration of neurofilament light chains (Nf-L) in a sample from the subject; and / or 4) measuring the concentration of soluble trigger receptor 2 (sTREM2) expressed on myeloid cells in a sample from the subject.

Description

[0001] This divisional application of the present invention is based on the patent application for invention with the application date of December 10, 2019, application number 201980087591.1 (international application number PCT / US2019 / 065485), and title "Biomarkers for Lysosomal Storage Disorders and Methods of Use Thereof".

[0002] Cross-reference to related applications

[0003] This application claims priority to U.S. Provisional Application Serial No. 62 / 777,599, filed on December 10, 2018; U.S. Provisional Application Serial No. 62 / 860,039, filed on June 11, 2019; U.S. Provisional Application Serial No. 62 / 869,387, filed on July 1, 2019; and U.S. Provisional Application Serial No. 62 / 912,253, filed on October 8, 2019. The entire contents of the applications cited above are incorporated herein by reference. Background of the Invention

[0004] Lysosomal storage disorders (LSDs) are relatively rare inherited metabolic diseases caused by lysosomal dysfunction. LSDs are typically caused by the deficiency of a single enzyme involved in the breakdown of metabolites in lysosomes. The accumulation of products due to the lack of enzyme activity affects various organ systems and can lead to severe symptoms and premature death. Most LSDs also have a significant neurological component, ranging from progressive neurodegeneration and severe cognitive impairment to epilepsy, behavioral, and psychiatric disorders. Despite extensive research to study the underlying molecular mechanisms of LSDs and develop new treatment methods, additional work is still needed. Specifically, new LSD biomarkers are needed for, e.g., assessing patients and therapies and for screening therapeutic agents. Summary of the Invention

[0005] Accordingly, certain embodiments described herein provide a method of detecting one or more biomarkers in a subject having a lysosomal storage disorder (LSD), the method comprising:

[0006] 1) measuring the concentration of a combination of two or more lipids in a sample from the subject, wherein the combination of lipids is selected from the group consisting of:

[0007] a) bis(monoacylglycerol) phosphate (BMP);

[0008] b) GM2 ganglioside and / or GM3 ganglioside;

[0009] c) GD3 ganglioside;

[0010] d) GD1a / b gangliosides; and

[0011] e) Glucosylceramide (GlcCer);

[0012] 2) Measuring the concentration of GlcCer in a sample from the subject, provided that the LSD is a mucopolysaccharidosis (MPS) disorder;

[0013] 3) Measuring the concentration of neurofilament light chain (Nf-L) in a sample from the subject; and / or

[0014] 4) Measuring the concentration of soluble triggering receptor expressed on myeloid cells 2 (sTREM2) in a sample from the subject.

[0015] Certain other embodiments of the invention provide a method of treating an LSD in a subject, the method comprising:

[0016] 1) Administering an LSD treatment to the subject;

[0017] 2) Measuring the concentration of:

[0018] a) A combination of two or more lipids in a sample from the subject, wherein the lipid combination is selected from the group consisting of:

[0019] i) BMP;

[0020] ii) GM2 ganglioside and / or GM3 ganglioside;

[0021] iii) GD3;

[0022] iv) GD1a / b; and

[0023] v) GlcCer;

[0024] b) GlcCer in a sample from the subject, provided that the LSD is an MPS disorder;

[0025] c) Nf-L in a sample from the subject; and / or

[0026] d) sTREM2 in a sample from the subject; and

[0027] 3) Adjusting the dose of the LSD treatment based on the concentration of the selected lipid / protein in the sample from the subject as compared to a control value.

[0028] Specifically, the invention includes but is not limited to the following:

[0029] 1. A method for detecting one or more biomarkers in a subject with lysosomal storage disease (LSD), the method comprising:

[0030] 1) Measuring the concentration of a combination of two or more lipids in a sample from the subject, wherein the combination of lipids is selected from the group consisting of:

[0031] a) Bis(monoacylglycerol) phosphate (BMP);

[0032] b) GM2 ganglioside and / or GM3 ganglioside;

[0033] c) GD3 ganglioside;

[0034] d) GD1a / b gangliosides; and

[0035] e) Glucosylceramide (GlcCer);

[0036] 2) Measuring the concentration of GlcCer in a sample from the subject, provided that the LSD is a mucopolysaccharidosis (MPS) disorder;

[0037] 3) Measuring the concentration of neurofilament light chain (Nf-L) in a sample from the subject; and / or

[0038] 4) Measuring the concentration of soluble triggering receptor expressed on myeloid cells 2 (sTREM2) in a sample from the subject.

[0039] 2. A method for evaluating the therapeutic efficacy in a subject with LSD, the method comprising:

[0040] 1) Measuring the concentration of a combination of two or more lipids in a sample obtained from the subject after administration of the treatment, wherein the combination of lipids is selected from the group consisting of:

[0041] a) BMP;

[0042] b) GM2 ganglioside and / or GM3 ganglioside;

[0043] c) GD3;

[0044] d) GD1a / b; and

[0045] e) GlcCer;

[0046] 2) Measuring the concentration of GlcCer in a sample obtained from the subject after administration of the treatment, provided that the LSD is an MPS disorder;

[0047] 3) Measure the concentration of Nf-L in a sample obtained from the subject after administering the treatment; and / or

[0048] 4) Measure the concentration of sTREM2 in a sample obtained from the subject after administering the treatment;

[0049] wherein a decrease in the concentration of the lipid / protein in the sample obtained from the subject after administering the treatment, compared to the concentration of the selected lipid / protein in a sample obtained from the subject before administering the treatment, is associated with treatment efficacy.

[0050] 3. The method according to item 1 or 2, further comprising administering an LSD treatment to the subject.

[0051] 4. The method according to any one of items 1-3, further comprising adjusting the treatment regimen of the subject.

[0052] 5. A method of treating an LSD in a subject, the method comprising:

[0053] 1) Administering an LSD treatment to the subject;

[0054] 2) Measuring the concentration of:

[0055] a) A combination of two or more lipids in a sample from the subject, wherein the combination of lipids is selected from the group consisting of:

[0056] i) BMP;

[0057] ii) GM2 ganglioside and / or GM3 ganglioside;

[0058] iii) GD3;

[0059] iv) GD1a / b; and

[0060] v) GlcCer;

[0061] b) GlcCer in a sample from the subject, provided that the LSD is an MPS disorder;

[0062] c) Nf-L in a sample from the subject; and / or

[0063] d) sTREM2 in a sample from the subject; and

[0064] 3) Adjusting the dose of the LSD treatment based on the concentration of the selected lipid / protein in the sample from the subject compared to a control value.

[0065] 6. The method according to any one of items 1 - 5, the method comprising measuring the concentration of a combination of two or more lipids.

[0066] 7. The method according to any one of items 1 - 5, the method comprising measuring the concentration of sTREM2.

[0067] 8. The method according to any one of items 1 - 5, the method comprising measuring the concentration of Nf - L.

[0068] 9. The method according to any one of items 1 - 5, the method comprising measuring the concentration of GlcCer, wherein the LSD is an MPS disorder.

[0069] 10. The method according to any one of items 1 - 5, the method comprising measuring the concentration of one or more lipids and the concentration of sTREM2.

[0070] 11. The method according to any one of items 1 - 5, the method comprising measuring the concentration of one or more lipids and the concentration of Nf - L.

[0071] 12. The method according to any one of items 1 - 6, wherein the combination comprises BMP.

[0072] 13. The method according to any one of items 1 - 6, wherein the combination comprises GlcCer.

[0073] 14. The method according to any one of items 1 - 6, wherein the combination comprises GD3.

[0074] 15. The method according to any one of items 1 - 6, wherein the combination comprises GD1a / b.

[0075] 16. The method according to any one of items 1 - 6, wherein the combination comprises GM2.

[0076] 17. The method according to any one of items 1 - 6, wherein the combination comprises GM3.

[0077] 18. The method according to any one of items 1-6, wherein the combination comprises: BMP and GlcCer; BMP and GD3; BMP and GD1a / b; BMP and GM2; BMP and GM3; GlcCer and GD3; GlcCer and GD1a / b; GlcCer and GM2; GlcCer and GM3; GD3 and GD1a / b; GD3 and GM2; GD3 and GM3; GD1a / b and GM2; GD1a / b and GM3; BMP, GlcCer and GD3; BMP, GlcCer and GD1a / b; BMP, GlcCer and GM2; BMP, GlcCer and GM3; BMP, GD3 and GD1a / b; BMP, GD3 and GM2; BMP, GD3 and GM3; BMP, GD1a / b and GM2; BMP, GD1a / b and GM3; BMP, GM2 and GM3; GlcCer, GD3 and GD1a / b; GlcCer, GD3 and GM2; GlcCer, GD3 and GM3; GlcCer, GD1a / b and GM2; GlcCer, GD1a / b and GM3; GlcCer, GM2 and GM3; GD3, GD1a / b and GM2; GD3, GD1a / b and GM3; GD3, GM2 and GM3; GD1a / b, GM2 and GM3; BMP, GlcCer, GD3 and GD1a / b; BMP, GlcCer, GD3 and GM2; BMP, GlcCer, GD3 and GM3; BMP, GlcCer, GD1a / b and GM2; BMP, GlcCer, GD1a / b and GM3; BMP, GlcCer, GM2 and GM3; BMP, GD3, GD1a / b and GM2; BMP, GD3, GD1a / b and GM3; BMP, GD3, GM2 and GM3; BMP, GD1a / b, GM2 and GM3; GlcCer, GD3, GD1a / b and GM2; GlcCer, GD3, GD1a / b and GM3; GlcCer, GD3, GM2 and GM3; GlcCer, GD1a / b, GM2 and GM3; GD3, GD1a / b, GM2 and GM3; BMP, GlcCer, GD3, GD1a / b and GM2; BMP, GlcCer, GD3, GD1a / b and GM3; BMP, GD3, GD1a / b, GM2 and GM3; BMP, GlcCer, GD3, GM2 and GM3; BMP, GlcCer, GD1a / b, GM2 and GM3; GlcCer, GD3, GD1a / b, GM2 and GM3; or BMP, GlcCer, GD3, GD1 / b, GM2 and GM3.

[0078] 19. The method according to any one of items 1 - 18, wherein the LSD is an MPS disorder.

[0079] 20. The method according to item 19, wherein the MPS disorder is Hunter syndrome.

[0080] 21. The method according to any one of items 2 - 20, wherein the LSD treatment comprises hematopoietic stem cell transplantation (HSCT), enzyme replacement therapy (ERT), substrate reduction therapy, chaperone therapy, and / or gene therapy.

[0081] 22. The method according to item 21, wherein the LSD treatment comprises brain - targeted ERT.

[0082] 23. The method according to item 22, wherein the LSD treatment is a protein comprising:

[0083] (a) a first Fc polypeptide linked to an enzyme replacement therapy (ERT) enzyme, an ERT enzyme variant, or a catalytically active fragment thereof; and

[0084] (b) a second Fc polypeptide that forms an Fc dimer with the first Fc polypeptide,

[0085] wherein the first Fc polypeptide and / or the second Fc polypeptide do not comprise immunoglobulin heavy and / or light chain variable region sequences or antigen - binding portions thereof.

[0086] 24. The method according to item 23, wherein the ERT enzyme is iduronate - 2 - sulfatase (IDS), an IDS variant, or a catalytically active fragment thereof.

[0087] 25. The method according to item 23, wherein the first Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 113, 193, and 197, and the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 114.

[0088] 26. The method according to item 23, wherein the first Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 113, 193, and 197, and the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 131.

[0089] 27. The method according to item 23, wherein the first Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 113, 193, and 197, and the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 167.

[0090] 28. The method according to item 23, wherein the first Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 113, 193, and 197, and the second Fc polypeptide comprises the amino acid sequence SEQ ID NO: 190.

[0091] 29. The method according to item 23, wherein the first Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 113, 193, and 197, and the second Fc polypeptide comprises the amino acid sequence SEQ ID NO: 191.

[0092] 30. The method according to item 23, wherein the first Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 113, 193, and 197, and the second Fc polypeptide comprises the amino acid sequence SEQ ID NO: 117.

[0093] 31. A method for active screening of a test agent for LSD treatment, the method comprising:

[0094] 1) contacting a cell with the test agent, wherein the lysosomal storage of the cell is impaired; and

[0095] 2) measuring the concentration of:

[0096] a) a combination of two or more lipids in the cell, wherein the lipid combination is selected from the group consisting of:

[0097] i) BMP;

[0098] ii) GM2 ganglioside and / or GM3 ganglioside;

[0099] iii) GD3;

[0100] iv) GD1a / b; and

[0101] v) GlcCer;

[0102] b) GlcCer in the cell, provided that the test agent is actively screened for MPS treatment;

[0103] c) Nf-L in the cell; and / or

[0104] d) sTREM2 in the cell;

[0105] wherein a decrease in the concentration of the selected lipid / protein in the cell compared to the concentration of the corresponding lipid / protein in control cells indicates that the test agent has activity as an LSD treatment.

[0106] 32. The method according to item 31, the method comprising measuring the concentration of sTREM2.

[0107] 33. The method according to item 31, the method comprising measuring the concentration of Nf-L.

[0108] 34. The method according to item 31, the method comprising measuring the concentration of GlcCer.

[0109] 35. The method according to item 31, the method comprising measuring the concentration of a combination of two or more lipids.

[0110] 36. The method according to item 31, the method comprising measuring the concentration of one or more lipids and the concentration of sTREM2.

[0111] 37. The method according to item 31, the method comprising measuring the concentration of one or more lipids and the concentration of Nf-L.

[0112] 38. The method according to any one of items 31-37, wherein the cell is a brain cell.

[0113] 39. A corrected CNS cell, the CNS cell comprising a reduced accumulation of metabolites, wherein a CNS cell lacking a lysosomal enzyme that causes the accumulation of the metabolite within the cell is contacted with a protein comprising:

[0114] (i) a first Fc polypeptide, which is linked to the lysosomal enzyme; and

[0115] (ii) a second Fc polypeptide, which forms an Fc dimer with the first Fc polypeptide, wherein the protein is capable of binding to the transferrin receptor (TfR),

[0116] to provide the corrected CNS cell comprising a reduced accumulation of the metabolite.

[0117] 40. The CNS cell according to item 39, wherein the protein binds to TfR with an affinity of about 50 nM to about 350 nM.

[0118] 41. The CNS cell according to item 39 or 40, wherein the enzyme is iduronate-2-sulfatase (IDS) or a catalytically active variant thereof.

[0119] 42. The CNS cell according to any one of items 39-41, wherein the metabolite is a glycosaminoglycan (GAG) and / or a lysosomal lipid selected from the group consisting of ganglioside, glucosylceramide, galactosylceramide, and BMP.

[0120] 43. The CNS cells as described in any one of items 39 - 42, wherein the CNS cells are selected from the group consisting of: neurons, astrocytes, and microglia.

[0121] 44. A method for sorting a population of CNS cells from a tissue sample, the method comprising:

[0122] (a) contacting the tissue sample with a primary antibody against a neuronal marker, a primary antibody against an astrocyte marker, a primary antibody against a microglia marker, a primary antibody against an endothelial marker, and a primary antibody against an oligodendrocyte marker, wherein each primary antibody is uniquely labeled to provide a labeled tissue sample; and

[0123] (b) sorting the cells in the labeled tissue sample by flow cytometry,

[0124] wherein the method provides unique cell populations of neurons, astrocytes, and microglia.

[0125] 45. The method as described in item 44, wherein the primary antibody against the neuronal marker is an anti - Thy1 antibody.

[0126] 46. The method as described in item 44 or 45, wherein the primary antibody against the microglia marker is an anti - CD11b antibody.

[0127] 47. The method as described in any one of items 44 - 46, wherein the primary antibody against the astrocyte marker is selected from the group consisting of an anti - EAAT2 antibody and an anti - astrocyte surface antigen - 2 (ACSA - 2) antibody.

[0128] 48. The method as described in any one of items 44 - 47, wherein the primary antibody against the endothelial marker is an anti - CD31 antibody.

[0129] 49. The method as described in any one of items 44 - 48, wherein the primary antibody against the oligodendrocyte marker is an anti - O1 antibody.

[0130] 50. The method as described in any one of items 44 - 49, the method providing a unique population of microglia comprising less than about 20% non - microglial cells, a unique population of astrocytes comprising less than about 20% non - astrocyte cells, and / or a unique population of neurons comprising less than about 20% non - neuronal cells.

[0131] 51. The method as described in any one of items 44 - 50, wherein the microglia population is sorted based on the marker profile O1 - / CD31 - / CD11b + sorting; the astrocyte population is based on the marker profile O1 - / CD31- / Thy1 - / EAAT2 + or O1 - / CD31 - / Thy1 - / ACSA-2 + Sorting; and / or said neuronal population based on the marker profile O1 - / CD31 - / Thy1 + / EAAT2 - or O1 - / CD31 - / Thy1 + / ACSA-2 - Sorting.

[0132] 52. The method according to any one of items 44-51, wherein the enriched cell population is analyzed to quantify sTREM2, Nf-L, metabolite and / or nucleic acid substance.

[0133] 53. The method according to item 52, wherein the metabolite is a glycosaminoglycan (GAG) substance or a lipid substance selected from the group consisting of ganglioside, glucosylceramide, galactosylceramide, and BMP.

[0134] 54. The method according to any one of items 44-53, wherein the enriched cell population is analyzed to quantify the administered therapeutic agent.

[0135] 55. The method according to item 54, wherein the administered therapeutic agent is ETV:IDS. Description of the Drawings

[0136] Figure 1 . Brain HS / DS (GAG) accumulation in IDS KO mice relative to age-matched controls. For each age group, wild type (WT) is shown on the left and IDS KO is shown on the right.

[0137] Figures 2A - 2D . Lysosomal lipids (GM2, GM3, BMP, and GlcCer) accumulate in the brains of IDS KO mice relative to age-matched controls. In Figures 2B - 2D , for each age group, the bar representing WT is shown on the left and the bar representing IDS KO is shown on the right.

[0138] Figure 3 . An elevated level of BMP is observed in the sera of IDS KO mice relative to age-matched controls. For each age group, the bar representing wild type (WT) is shown on the left and the bar representing IDS KO is shown on the right.

[0139] Figure 4 . Elevated levels of lysosomal lipids (Gd1a / b, GM3, BMP, and GlcCer) were observed in the CSF of IDS KO mice relative to age-matched controls. For each age group, the bars representing wild-type (WT) are shown on the left, and the bars representing IDS KO are shown on the right.

[0140] Figures 5A - 5B . Peripheral administration of ETV:IDS (4-week treatment) corrected the ( Figure 5A ) brain and ( Figure 5B ) CSF GAG accumulation in IDS KO mice. The graphs show mean ± 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.

[0141] Figure 6 . Peripheral administration of ETV:IDS (4-week treatment) corrected the accumulation of lysosomal lipids (gangliosides) in the brains of IDS KO mice. The graphs show mean ± 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.

[0142] Figures 7A - 7B . Peripheral administration of ETV:IDS (4-week treatment) corrected the accumulation of lysosomal lipids (GlcCer) in the brains of IDS KO mice. The graphs show mean ± 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.

[0143] Figure 8 . Peripheral administration of ETV:IDS (4-week treatment) corrected the accumulation of lysosomal lipids (BMP) in the brains of IDS KO mice. The graphs show mean ± 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.

[0144] Figure 9 . Heat map showing lipid levels in the brains of IDS KO mice treated with vehicle, ETV:IDS, or Elaprase (idursulfase). Fold change is relative to WT.

[0145] Figure 10.ETV:IDS peripheral administration corrected TREM2 accumulation in IDS KO brains. Graphs show mean ± SEM and p values: one-way ANOVA with Dunnett's multiple comparison test; ****p < 0.0001.

[0146] Figure 11 .ETV:IDS peripheral administration decreased IDS KO; TfR mu / hu accumulation of CSF soluble TREM2 (sTrem2) levels in KI mice.

[0147] Figures 12A - 12C . Serum ( Figure 12A ), brain ( Figure 12B ), and CSF ( Figure 12C ) HS / DS (GAG) accumulation in IDS KO mice treated with vehicle or 3, 10, 20, or 40 mg / kg ETV:IDS.

[0148] Figures 13A - 13C . Peripheral administration of ETV:IDS corrected GM3 ( Figure 13A ), GlcCer ( Figure 13B ), and BMP ( Figure 13C ) accumulation in the brains of IDS KO mice treated with 3, 10, 20, or 40 mg / kg ETV:IDS compared to vehicle-treated IDS KO mice. Graphs show mean ± 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.

[0149] Figures 14A - 14B . Administration of ETV:IDS decreased the levels of individual GAG species (D0S0, D0A0, and D0a4) in the brain ( Figure 14A ) and CSF ( Figure 14B ) from IDS KO mice compared to vehicle-treated IDS KO mice. Graphs show mean ± SEM and p values: one-way ANOVA with Dunnett's multiple comparison test; **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

[0150] Figures 15A - 15C . Figure 15A Schematic of the CNS cell sorting protocol for isolating enriched populations of neurons, astrocytes, and microglia. Figure 15B Flow chart of the representative gating protocol for isolating enriched populations. Figure 15C Shows Figure 15BRepresentative FACS gating of the sorting procedure described. Starting from the upper left to the lower right: Forward (FSC) and side (SSC) scatter determine cells in debris; live cell main gating; confirmation of exclusion of CD31-positive endothelial cells; EAAT2-positive astrocytes subgated from CD11b microglia; Thy1-positive neurons subgated from EAAT2-positive astrocytes; and finally, removal of O1 oligodendrocytes from the CD11b microglia, EAAT2 astrocytes, and Thy1 neuron cell populations determined the final sorting criteria.

[0151] Figure 16 . Principal component analysis was performed using log-transformed CPM expression values from the top 500 genes with the highest variance. Principal components 1 and 2 accounted for 48% and 26% of the variance between samples, respectively.

[0152] Figures 17A - 17C . Figure 17A Shows the expression of certain cell-specific markers in purified neurons, astrocytes, microglia, and input cell suspensions determined by RNA-Seq. Rows are genomic groups specific to cell types: (endo.) endothelial, (oligo.) oligodendrocyte; the expression value of each gene is depicted as the number of standard deviations from its mean (z-score); n = 4 mice. Figure 17B Lists and shows the expression of the top 20 enriched genes determined by an enrichment fold >5.0 and FDR <0.01 for each cell type. Heatmap expression values were plotted as gene-level z-scores. Input cells were single-cell suspensions from dissociated brains. Figure 17C Shows qRT-PCR data generated from isolated cell populations of neurons, astrocytes, and microglia. Gene categories are grouped on the x-axis: (Astro) astrocytes, (MG) microglia, (Neu) neurons, (Oligo) oligodendrocytes, and (Endo) endothelial cells; n = 5 mice. The graph shows mean ± SEM.

[0153] Figure 18A . From IDS KO; TfR mu / hu GAG levels in microglia, astrocytes, and neurons isolated from KI mice, relative to TfR mu / hu KI controls (n = 3 - 5 mice per group). All data are shown as mean ± SEM; unpaired student’s t-test p ≤ 0.05*, 0.001***.

[0154] Figure 18B . In vehicle-treated TfR mu / huKI and IDS KO; TfR treated with ETV:IDS (40 mg / kg) mu / hu Distribution of ETV:IDS in neuron, astrocyte, and microglia cell populations enriched by FACS 2 hours after dosing in KI mice; n = 4 mice per group. Graphs show mean ± SEM; two-way ANOVA with Sidak’s test; **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

[0155] Figure 19 . From IDS KO; TfR mu / hu Reduced GAG accumulation in neurons, astrocytes, and microglia of IDS KO; TfR KI mice, with mice intravenously administered 40 mg / kg ETV:IDS once a week for four weeks. Data are shown as mean ± SEM; one-way ANOVA with Tukey′s multiple comparison test; *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.

[0156] Figure 20 . From IDS KO; TfR mu / hu Reduced ganglioside accumulation in neurons, astrocytes, and microglia of IDS KO; TfR KI mice, with mice intravenously administered 40 mg / kg ETV:IDS once a week for four weeks. Data are shown as mean ± SEM; one-way ANOVA with Tukey′s multiple comparison test; *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.

[0157] Figure 21 . From IDS KO; TfR mu / hu Reduced glucosylceramide accumulation in neurons, astrocytes, and microglia of IDS KO; TfR KI mice, with mice intravenously administered 40 mg / kg ETV:IDS once a week for four weeks. Data are shown as mean ± SEM; one-way ANOVA with Tukey′s multiple comparison test; *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.

[0158] Figure 22 . From IDS KO; TfR mu / hu Amelioration of BMP alterations in neurons, astrocytes, and microglia of IDS KO; TfR KI mice, with mice intravenously administered 40 mg / kg ETV:IDS once a week for four weeks. Data are shown as mean ± SEM; one-way ANOVA with Tukey′s multiple comparison test; *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.

[0159] Figure 23 . After weekly administration of vehicle, idursulfase, or ETV:IDS for four weeks, wild-type and IDS KO;TfR mu / hu H&E staining (top panel) and MALDI MS images (middle and bottom panels) of coronal brain sections from KI mice. The middle panel shows signal distribution at m / z 1382.816, corresponding to GM2 (d36:1); and the bottom panel shows signal distribution at m / z 1179.738, corresponding to GM3 (d36:1). The images depict the relative intensity of each signal from 0 - 100%.

[0160] Figure 24 . From TfR mu / hu KI and IDS KO;TfR mu / hu Immunofluorescence images of brain tissues from KI mice and IDS KO;TfR KI mice treated with vehicle, ETV:IDS, or idursulfase. The brain tissues were stained for DAPI and CD68. The magnification is 20x, and the images represent the hippocampus (top panel), cortex (middle panel), and striatum (bottom panel).

[0161] Figure 25 . Table of cell type-specific enriched gene sets; the information in the table corresponds to the Figure 17B heatmap. Expression of the top 20 genes determined by enrichment fold >5.0 and FDR <0.01 for each cell type is listed in ascending order of p-value. The average log fold change (logFC.avg) is relative to two other "outgroup" populations, and the rightmost three columns show the average expression of genes within each cell population; n = 4 mice.

[0162] Figures 26A - 26C . Scatter plot of Nf-L concentration in 3-month-old, 6-month-old, and 9-month-old wild-type and IDS knockout mice. Figure 26A and Figure 26B show the Nf-L levels in serum and CSF of 9-month-old mice, respectively. Figure 26C shows the Nf-L levels in CSF of mouse cohorts of different ages. Data are shown as mean + / - SEM, and p-values were obtained by unpaired t-test analysis.

[0163] Figure 27 . Peripheral administration of ETV:IDS (1 mg / kg or 3 mg / kg given weekly for 13 weeks) reduces the neurofilament light chain (Nf-L) level in IDS knockout mice. Data are shown as mean + / - SEM, and p-values were obtained by unpaired t-test analysis (*p < 0.05; ***p < 0.001; error bars = SEM).

[0164] Figure 28.ETV: IDS peripheral administration (1 mg / kg or 3 mg / kg once a week for 13 weeks) reduced the accumulation of GAG levels in the liver and urine of IDS knockout mice. Urinary GAG levels were normalized relative to creatinine. Data are shown as mean + / - SEM, and p-values were obtained by unpaired t-test analysis (****p < 0.0001; error bars = SEM). Detailed Description

[0165] As described herein, a series of biomarkers associated with LSD have been identified. Specifically, as described in the Examples, a series of secondary lysosomal lipids were shown to accumulate in the brain, CSF, and serum from LSD mouse models. In addition, triggering receptor 2 (TREM2) and neurofilament light chain (Nf-L), which are expressed on myeloid cells, were shown to accumulate in the brain tissue from these mice. Notably, the accumulation of the secondary lipids and TREM2 was ameliorated by administration of LSD treatment (e.g., ETV:IDS). Based on these findings, these lipids / proteins can be used as biomarkers, including but not limited to, in methods for assessing a subject having such a disorder, assessing the efficacy of certain treatments, developing and / or modifying treatment regimens (e.g., adjusting dosages), and screening for therapeutic agents.

[0166] Accordingly, certain embodiments described herein provide a method for detecting one or more biomarkers in a subject having an LSD, the method comprising:

[0167] 1) measuring the concentration of a combination of two or more lipids in a sample from the subject, wherein the combination of lipids is selected from the group consisting of:

[0168] a) BMP;

[0169] b) GM2 ganglioside and / or GM3 ganglioside;

[0170] c) GD3 ganglioside;

[0171] d) GD1a / b ganglioside; and

[0172] e) GlcCer;

[0173] 2) measuring the concentration of GlcCer in a sample from the subject, provided that the LSD is an MPS disorder; and / or

[0174] 3) measuring the concentration of sTREM2 in a sample from the subject.

[0175] Certain embodiments described herein also provide a method for detecting one or more biomarkers in a subject having an LSD, the method comprising:

[0176] 1) Measure the concentration of a combination of two or more lipids in a sample from the subject, wherein the combination of lipids is selected from the group consisting of:

[0177] a) BMP;

[0178] b) GM2 ganglioside and / or GM3 ganglioside;

[0179] c) GD3 ganglioside;

[0180] d) GD1a / b ganglioside; and

[0181] e) GlcCer;

[0182] 2) Measure the concentration of GlcCer in a sample from the subject, provided that the LSD is an MPS disorder;

[0183] 3) Measure the concentration of Nf-L in a sample from the subject; and / or

[0184] 4) Measure the concentration of sTREM2 in a sample from the subject.

[0185] Certain embodiments described herein also provide a method for assessing the therapeutic efficacy in a subject with an LSD, the method comprising:

[0186] 1) Measure the concentration of a combination of two or more lipids in a sample from the subject (i.e., a sample obtained from the subject after administration of the treatment), wherein the combination of lipids is selected from the group consisting of:

[0187] a) BMP;

[0188] b) GM2 ganglioside and / or GM3 ganglioside;

[0189] c) GD3 ganglioside;

[0190] d) GD1a / b ganglioside; and

[0191] e) GlcCer;

[0192] 2) Measure the concentration of GlcCer in a sample from the subject, provided that the LSD is an MPS disorder; and / or

[0193] 3) Measure the concentration of sTREM2 in a sample from the subject;

[0194] wherein a decrease in the concentration of the lipid / protein in the sample from the subject, as compared to the concentration of the selected lipid / protein in a sample obtained from the subject prior to administration of the treatment, is associated with treatment efficacy.

[0195] Certain embodiments described herein provide a method of assessing treatment efficacy in a subject with an LSD, the method comprising:

[0196] 1) measuring the concentration of a combination of two or more lipids in a sample from the subject (i.e., a sample obtained from the subject after administration of the treatment), wherein the combination of lipids is selected from the group consisting of:

[0197] a) BMP;

[0198] b) GM2 ganglioside and / or GM3 ganglioside;

[0199] c) GD3 ganglioside;

[0200] d) GD1a / b ganglioside; and

[0201] e) GlcCer;

[0202] 2) measuring the concentration of GlcCer in a sample from the subject, provided that the LSD is an MPS disorder;

[0203] 3) measuring the concentration of Nf-L in a sample from the subject; and / or

[0204] 4) measuring the concentration of sTREM2 in a sample from the subject;

[0205] wherein a decrease in the concentration of the lipid / protein in the sample from the subject, as compared to the concentration of the selected lipid / protein in a sample obtained from the subject prior to administration of the treatment, is associated with treatment efficacy.

[0206] Certain embodiments described herein provide a method of identifying a subject with an LSD as a treatment candidate, the method comprising:

[0207] 1) measuring the concentration of a combination of two or more lipids in a sample from the subject, wherein the combination of lipids is selected from the group consisting of:

[0208] a) BMP;

[0209] b) GM2 ganglioside and / or GM3 ganglioside;

[0210] c) GD3 ganglioside;

[0211] d) GD1a / b ganglioside; and

[0212] e) GlcCer;

[0213] 2) Measuring the concentration of GlcCer in a sample from the subject, provided that the LSD is an MPS disorder; and / or

[0214] 3) Measuring the concentration of sTREM2 in a sample from the subject;

[0215] wherein the subject is identified as a treatment candidate or a non-treatment candidate based on the concentration of the selected lipid / protein in the sample compared to a control value. For example, the subject is identified as a treatment candidate if the concentration of the selected lipid / protein in the sample from the subject is at least as high as the control value.

[0216] Certain embodiments described herein also provide a method of identifying a subject with an LSD as a treatment candidate, the method comprising:

[0217] 1) Measuring the concentration of a combination of two or more lipids in a sample from the subject, wherein the combination of lipids is selected from the group consisting of:

[0218] a) BMP;

[0219] b) GM2 ganglioside and / or GM3 ganglioside;

[0220] c) GD3 ganglioside;

[0221] d) GD1a / b ganglioside; and

[0222] e) GlcCer;

[0223] 2) Measuring the concentration of GlcCer in a sample from the subject, provided that the LSD is an MPS disorder;

[0224] 3) Measuring the concentration of Nf-L in a sample from the subject; and / or

[0225] 4) Measuring the concentration of sTREM2 in a sample from the subject;

[0226] wherein the subject is identified as a treatment candidate or a non-treatment candidate based on the concentration of the selected lipid / protein in the sample compared to a control value. For example, the subject is identified as a treatment candidate if the concentration of the selected lipid / protein in the sample from the subject is at least as high as the control value.

[0227] In certain embodiments, the methods described herein further comprise administering an LSD treatment to a subject. In certain embodiments, the methods described herein further comprise adjusting the treatment regimen of the subject. For example, based on a comparison of the concentration of each of the selected lipids / proteins to a control value, the dosage may be increased or decreased, the dosing frequency may be increased or decreased, or an alternative therapy may be administered.

[0228] Accordingly, certain embodiments described herein provide a method of treating an LSD in a subject, the method comprising:

[0229] 1) administering an LSD treatment to the subject;

[0230] 2) measuring the concentration of:

[0231] a) a combination of two or more lipids in a sample from the subject, wherein the combination of lipids is selected from the group consisting of:

[0232] i) BMP;

[0233] ii) GM2 ganglioside and / or GM3 ganglioside;

[0234] iii) GD3 ganglioside;

[0235] iv) GD1a / b ganglioside; and

[0236] v) GlcCer;

[0237] b) GlcCer in a sample from the subject, provided that the LSD is an MPS disorder; and / or

[0238] c) sTREM2 in a sample from the subject; and

[0239] 3) adjusting the dosage of the LSD treatment based on the concentration of the selected lipids / proteins in the sample from the subject as compared to a control value. In certain embodiments, the method comprises administering an adjusted dosage of the LSD treatment to the subject, wherein the dosage adjustment is based on the concentration of the selected lipids / proteins as compared to a control value. In certain embodiments, the method comprises administering an LSD treatment to the subject at a dosage higher than the original LSD treatment dosage (step 1) when the concentration of the selected lipids / proteins is higher than the control value. In certain embodiments, the method comprises administering an LSD treatment to the subject at a dosage lower than the original LSD treatment dosage (step 1) when the concentration of the selected lipids / proteins is lower than the control value.

[0240] Certain embodiments described herein further provide a method of treating an LSD in a subject, the method comprising:

[0241] 1) Administer an LSD treatment to the subject;

[0242] 2) Measure the concentration of:

[0243] a) A combination of two or more lipids in a sample from the subject, wherein the lipid combination is selected from the group consisting of:

[0244] i) BMP;

[0245] ii) GM2 ganglioside and / or GM3 ganglioside;

[0246] iii) GD3 ganglioside;

[0247] iv) GD1a / b ganglioside; and

[0248] v) GlcCer;

[0249] b) GlcCer in a sample from the subject, provided that the LSD is an MPS disorder;

[0250] c) Nf-L in a sample from the subject; and / or

[0251] d) sTREM2 in a sample from the subject; and

[0252] 3) Adjust the dose of the LSD treatment based on the concentration of the selected lipid / protein in the sample from the subject compared to a control value. In certain embodiments, the method includes administering an adjusted dose of the LSD treatment to the subject, wherein the dose adjustment is based on the concentration of the selected lipid / protein compared to a control value. In certain embodiments, the method includes administering an LSD treatment to the subject at a dose higher than the original LSD treatment dose (step 1) when the concentration of the selected lipid / protein is higher than the control value. In certain embodiments, the method includes administering an LSD treatment to the subject at a dose lower than the original LSD treatment dose (step 1) when the concentration of the selected lipid / protein is lower than the control value.

[0253] In certain embodiments, the methods described herein include measuring or having measured the concentration of sTREM2 in a sample from a subject with an LSD.

[0254] In certain embodiments, the methods described herein include measuring or having measured the concentration of Nf-L in a sample from a subject with an LSD.

[0255] As described herein, the concentration of GlcCer in a sample from a subject with LSD can be measured. In such an embodiment, the LSD is MPS. Thus, in certain embodiments, the methods described herein include measuring or having measured the concentration of GlcCer in a sample from a subject with LSD, provided that the LSD is MPS. In certain other embodiments, GlcCer is measured in combination with other lipids / proteins described herein. In such an embodiment, the LSD can be any LSD, such as the LSDs described herein.

[0256] In certain embodiments, the methods described herein include measuring or having measured the concentration of a combination of two or more lipids in a sample from a subject with LSD.

[0257] In certain embodiments, the methods described herein include measuring or having measured the concentration of a combination of one or more lipids and the concentration of sTREM2 in a sample from a subject with LSD.

[0258] In certain embodiments, the methods described herein include measuring or having measured the concentration of a combination of one or more lipids and the concentration of Nf-L in a sample from a subject with LSD.

[0259] In certain embodiments, the methods described herein include measuring or having measured the concentration of sTREM2 and the concentration of Nf-L in a sample from a subject with LSD.

[0260] In certain embodiments, the methods described herein include measuring or having measured the concentration of a combination of one or more lipids, the concentration of sTREM2, and the concentration of Nf-L in a sample from a subject with LSD.

[0261] Certain embodiments described herein provide a method of treating a subject with LSD, the method comprising administering an LSD treatment to the subject, wherein the subject has or has been determined to have:

[0262] 1) a combination of two or more lipids having an increased concentration compared to a control, wherein the combination of lipids is selected from the group consisting of:

[0263] a) BMP;

[0264] b) GM2 ganglioside and / or GM3 ganglioside;

[0265] c) GD3 ganglioside;

[0266] d) GD1a / b ganglioside; and

[0267] e) GlcCer;

[0268] 2) increased concentration of GlcCer, provided that the LSD is an MPS disorder; and / or

[0269] 3) increased concentration of sTREM2.

[0270] Certain embodiments described herein provide a method of treating an LSD in a subject, the method comprising administering an LSD treatment to the subject, wherein the subject has or has been determined to have:

[0271] 1) a combination of two or more lipids with increased concentration compared to a control, wherein the combination of lipids is selected from the group consisting of:

[0272] a) BMP;

[0273] b) GM2 ganglioside and / or GM3 ganglioside;

[0274] c) GD3 ganglioside;

[0275] d) GD1a / b ganglioside; and

[0276] e) GlcCer;

[0277] 2) increased concentration of GlcCer, provided that the LSD is an MPS disorder;

[0278] 3) increased concentration of Nf-L; and / or

[0279] 4) increased concentration of sTREM2.

[0280] Certain embodiments described herein provide a method of treating an LSD in a subject, the method comprising:

[0281] 1) obtaining or having obtained a sample from the subject;

[0282] 2) detecting or having detected in the sample:

[0283] a) an increase in the concentration of a combination of two or more lipids in the sample compared to a control, wherein the combination of lipid / protein is selected from the group consisting of:

[0284] i) BMP;

[0285] ii) GM2 ganglioside and / or GM3 ganglioside;

[0286] iii) GD3 ganglioside;

[0287] iv) GD1a / b ganglioside; and

[0288] v) GlcCer;

[0289] b) The concentration of GlcCer in the sample is increased compared to a control, provided that the LSD is an MPS disorder; and / or

[0290] c) The concentration of sTREM2 in the sample is increased compared to a control;

[0291] 3) When an increased concentration of the selected lipid / protein is detected, diagnose the subject as having an LSD; and

[0292] 4) Administer an effective amount of LSD treatment to the diagnosed subject.

[0293] Certain embodiments described herein provide a method of treating an LSD in a subject, the method comprising:

[0294] 1) Obtain or have obtained a sample from the subject;

[0295] 2) Detect or have detected in the sample that:

[0296] a) The concentration of a combination of two or more lipids in the sample is increased compared to a control, wherein the lipid / protein combination is selected from the group consisting of:

[0297] i) BMP;

[0298] ii) GM2 ganglioside and / or GM3 ganglioside;

[0299] iii) GD3 ganglioside;

[0300] iv) GD1a / b ganglioside; and

[0301] v) GlcCer;

[0302] b) The concentration of GlcCer in the sample is increased compared to a control, provided that the LSD is an MPS disorder;

[0303] c) The concentration of Nf-L in the sample is increased compared to a control; and / or

[0304] d) The concentration of sTREM2 in the sample is increased compared to a control.

[0305] 3) When an increased concentration of the selected lipid / protein is detected, diagnose the subject as having an LSD; and

[0306] 4) Administer an effective amount of LSD treatment to the diagnosed subject.

[0307] In certain embodiments, the subject has or is determined to have increased concentrations of sTREM2.

[0308] In certain embodiments, the subject has or is determined to have increased concentrations of Nf-L.

[0309] In certain embodiments, the subject has or is determined to have increased concentrations of GlcCer.

[0310] In certain embodiments, the subject has or is determined to have increased concentrations of a combination of two or more lipids.

[0311] In certain embodiments, the subject has or is determined to have increased concentrations of one or more lipids and increased concentrations of sTREM2.

[0312] In certain embodiments, the subject has or is determined to have increased concentrations of one or more lipids and increased concentrations of Nf-L.

[0313] In certain embodiments, the subject has or is determined to have increased concentrations of Nf-L and increased concentrations of sTREM2.

[0314] In certain embodiments, the subject has or is determined to have increased concentrations of one or more lipids, increased concentrations of Nf-L, and increased concentrations of sTREM2.

[0315] Certain embodiments also provide LSD treatment for use in the methods described herein.

[0316] Certain embodiments provide the use of LSD treatment for the preparation of a medicament for use in the methods described herein.

[0317] Biomarkers of lysosomal storage diseases

[0318] As described herein, a series of LSD biomarkers have been identified. Specifically, these biomarkers include the accumulation of specific lipids (i.e., BMP, GlcCer, GD3, GD1a / b, GM2, and GM3) in subjects with LSD, as well as the accumulation of TREM2 (which can be measured based on sTREM2 levels) and the accumulation of Nf-L. Thus, these biomarkers can be evaluated by measuring the concentration of one or more of these lipids / proteins in a sample obtained from a subject.

[0319] As used herein, the phrase "sample" or "biological sample" means a biological sample obtained from a subject containing protein and / or lipid. Thus, the sample can be evaluated at the lipid or protein level. In certain embodiments, the biological sample comprises tissue, cerebrospinal fluid (CSF), urine, blood, serum, or plasma. In certain embodiments, the sample comprises tissue, such as brain, liver, kidney, lung, or spleen. The sample can include a fluid. In certain embodiments, the sample comprises CSF. In certain embodiments, the sample comprises blood and / or plasma. In certain embodiments, the sample comprises serum.

[0320] TREM2

[0321] As used herein, the term "TREM2 protein" refers to the triggering receptor 2 protein expressed on myeloid cells and encoded by the gene Trem2. As used herein, "TREM2 protein" refers to the native (i.e., wild-type) TREM2 protein of any vertebrate, such as (but not limited to) human, non-human primate (e.g., cynomolgus macaque), rodent (e.g., mouse, rat), and other mammals. In some embodiments, the TREM2 protein is the human TREM2 protein having the sequence identified in UniprotKB accession number Q9NZC2.

[0322] The TREM2 gene encodes a protein that is 230 amino acids in length and includes an extracellular domain, a transmembrane region, and a short cytoplasmic tail (see UniProtKB Q9NZC2; NCBI reference sequence: NP_061838.1). The extracellular region encoded by exon 2 consists of a single type of V Ig-SF domain and contains three potential N-glycosylation sites. The putative transmembrane region contains a charged lysine residue. The cytoplasmic tail of TREM2 lacks a signaling motif and is thought to signal through the signaling adaptor molecule DAP12 / TYROBP and through DAP10. TREM2 is found on the surface of osteoclasts, immature dendritic cells, and macrophages. In the central nervous system, TREM2 is expressed only in microglia.

[0323] TREM2 can be cleaved by a disintegrin and metalloproteinase (ADAM) protease (such as ADAM10 and ADAM17), thereby releasing soluble TREM2 (sTREM2) into the extracellular environment. As described herein, an increase in the level of TREM2 indicates downstream pathology in a subject with LSD. Thus, the levels of TREM2 or sTREM2 can be measured using assays known in the art or described herein. For example, assays for detecting and measuring protein expression levels include, for example, Western blot analysis, immunofluorescence, immunohistochemistry (such as tissue arrays), MesoScale Discovery (MSD) methods, and the like. In certain methods described herein, the concentration of TREM2 can be measured in a sample from a subject with or suspected of having LSD. In certain methods described herein, the concentration of sTREM2 can be measured in a sample from a subject with or suspected of having LSD.

[0324] In certain embodiments, the concentration of sTREM2 in a sample from a subject with LSD is increased compared to a control (such as a healthy control subject without LSD). In certain embodiments, the concentration of sTREM2 is increased by at least about 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more compared to the control. In certain embodiments, an increase in the concentration of sTREM2 is observed in a tissue sample such as the brain. In certain embodiments, an increase in the concentration of sTREM2 is observed in the CSF. In certain embodiments, an increase in the concentration of sTREM2 is observed in the serum.

[0325] In certain other embodiments, an effective LSD treatment is administered to a subject with LSD, which results in a decrease in the concentration of sTREM2 in a sample from the subject compared to a control (such as the same subject before receiving the treatment). In certain embodiments, the concentration of sTREM2 is decreased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more compared to the control. In certain embodiments, a decrease in the concentration of sTREM2 is observed in a tissue sample such as the brain. In certain embodiments, a decrease in the concentration of sTREM2 is observed in the CSF. In certain embodiments, a decrease in the concentration of sTREM2 is observed in the serum.

[0326] Nf-L

[0327] As used herein, the term "Nf-L" refers to neurofilament light chain encoded by the gene NEFL (also known as neurofilament light chain polypeptide, neurofilament light polypeptide, and neurofilament light protein). As used herein, "Nf-L protein" refers to the native (i.e., wild-type) Nf-L protein of any vertebrate, such as (but not limited to) human, non-human primate (such as cynomolgus macaque), rodent (such as mouse, rat), and other mammals. In some embodiments, the Nf-L protein is the human Nf-L protein having the sequence identified in UniprotKB accession number P07196.

[0328] As described herein, an increase in the level of Nf-L indicates downstream pathology in a subject with LSD. Accordingly, Nf-L levels can be measured using assays known in the art or described herein. By way of example, assays for detecting and measuring protein expression levels include, for example, Western blot analysis, immunofluorescence, immunohistochemistry (such as tissue arrays), MesoScale Discovery (MSD) methods, and the like. In certain methods described herein, the Nf-L concentration in a sample from a subject with or suspected of having LSD can be measured.

[0329] In certain embodiments, the Nf-L concentration in a sample from a subject with LSD is increased compared to a control (such as a healthy control subject without LSD). In certain embodiments, the Nf-L concentration is increased by at least about 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more compared to the control. In certain embodiments, an increase in the Nf-L concentration is observed in a tissue sample such as the brain. In certain embodiments, an increase in the Nf-L concentration is observed in the CSF. In certain embodiments, an increase in the Nf-L concentration is observed in the serum.

[0330] In certain other embodiments, an effective LSD treatment is administered to a subject with LSD, which results in a decrease in the concentration of Nf-L in a sample from the subject compared to a control (such as the same subject prior to receiving the treatment). In certain embodiments, the concentration of Nf-L is decreased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more compared to the control. In certain embodiments, a decrease in the Nf-L concentration is observed in a tissue sample such as the brain. In certain embodiments, a decrease in the Nf-L concentration is observed in the CSF. In certain embodiments, a decrease in the Nf-L concentration is observed in the serum.

[0331] Lipid

[0332] As described herein, an increase in the levels of BMP, GlcCer, GD3, GD1a / b, GM2, and / or GM3 indicates downstream pathology in a subject with an LSD. Thus, in certain methods described herein, the concentration of at least one of BMP, GlcCer, GD3, GD1a / b, GM2, and / or GM3 can be measured in a sample from a subject with or suspected of having an LSD. The concentration of these lipids can be measured using assays known in the art or described herein (e.g., by mass spectrometry).

[0333] Bis(monoacylglycerol)phosphate (BMP) refers to a class of anionic phospholipids. BMP is enriched in the inner membranes of multivesicular endosomes and lysosomes and is thought to play a role in glycosphingolipid degradation and cholesterol trafficking (see Kobayashi et al., Nat. Cell Biol. 1 (1999) 113-118). Specific BMP species are described herein (see, e.g., the Examples and Figures).

[0334] In certain embodiments, the concentration of at least one BMP species in a sample from a subject with an LSD is increased compared to a control (e.g., a healthy control subject without an LSD). In certain embodiments, the BMP is a BMP species described herein, e.g., in the Examples or Figures. By way of example, in certain embodiments, the BMP is BMP(44:12), BMP(36:2), BMP(di20:4), BMP(di22:6), or BMP(di18:1). In certain embodiments, the concentration of at least one BMP is increased by at least about 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more compared to a control. In certain embodiments, an increase in BMP concentration is observed in a tissue sample such as the brain. In certain embodiments, an increase in BMP concentration is observed in the CSF. In certain embodiments, an increase in BMP concentration is observed in the serum.

[0335] In certain other embodiments, an effective LSD treatment is administered to a subject with an LSD, which results in a decrease in the concentration of at least one BMP species in a sample from the subject compared to a control (e.g., the same subject prior to receiving the treatment). In certain embodiments, the concentration of at least one BMP is decreased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more compared to a control. In certain embodiments, a decrease in BMP concentration is observed in a tissue sample such as the brain. In certain embodiments, a decrease in BMP concentration is observed in the CSF. In certain embodiments, a decrease in BMP concentration is observed in the serum.

[0336] Glucosylceramide (GlcCer) is a glycosphingolipid (ceramide and oligosaccharide) or oligosylceramide with one or more sialic acids attached to the sugar chain. Specific GlcCer substances are described herein (see, e.g., the Examples and the Figures).

[0337] In certain embodiments, the concentration of at least one GlcCer substance in a sample from a subject with LSD is increased compared to a control (e.g., a healthy control subject without LSD). In certain embodiments, the GlcCer is a GlcCer substance as described herein, e.g., in the Examples or the Figures. By way of example, in certain embodiments, the GlcCer is GlcCer(d34:0), GlcCer(d34:1), GlcCer(d36:1), GlcCer(d42:1), GlcCer(d18:1,16:0), GlcCer(d18:1,18:0), GlcCer(d18:2,18:0), GlcCer(d18:1,20:0), GlcCer(d18:2,20:0), GlcCer(d18:1,22:0), GlcCer(d18:1,22:1), GlcCer(d18:2,22:0), GlcCer(d18:1,24:1), or GlcCer(d18:1,24:0). In certain embodiments, the GlcCer is GlcCer(d34:1), GlcCer(d36:1), GlcCer(d42:1), GlcCer(d18:1,16:0), or GlcCer(d18:1,22:0). In certain embodiments, the GlcCer is GlcCer(d34:0). In certain embodiments, the concentration of at least one GlcCer is increased by at least about 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more compared to the control. In certain embodiments, an increase in GlcCer concentration is observed in a tissue sample such as the brain. In certain embodiments, an increase in GlcCer concentration is observed in the CSF. In certain embodiments, an increase in GlcCer concentration is observed in the serum.

[0338] In certain other embodiments, an effective LSD treatment is administered to a subject having an LSD, which results in a decrease in the concentration of at least one GlcCer species in a sample from the subject as compared to a control (e.g., the same subject prior to receiving the treatment). In certain embodiments, the concentration of at least one GlcCer is decreased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more as compared to the control. In certain embodiments, a decrease in GlcCer concentration is observed in a tissue sample such as, for example, the brain. In certain embodiments, a decrease in GlcCer concentration is observed in the CSF. In certain embodiments, a decrease in GlcCer concentration is observed in the serum.

[0339] Gangliosides are a class of glycosphingolipids. Specific GD3 species are described herein (see, e.g., the Examples and the Figures).

[0340] In certain embodiments, the concentration of at least one GD3 species in a sample from a subject having an LSD is increased as compared to a control (e.g., a healthy control subject without LSD). In certain embodiments, GD3 is a GD3 species described herein, e.g., in the Examples or the Figures. By way of example, in certain embodiments, GD3 is GD3(d34:1), GD3(d36:1), GD3(d38:1), GD3(d39:1), GD3(d40:1), GD3(d42:2) or GD3(d42:1). In certain embodiments, GD3 is GD3(d34:1), GD3(d36:1) or GD3(d39:1). In certain embodiments, GD3 is GD3(d34:1) or GD3(d36:1).

[0341] In certain embodiments, the concentration of at least one GD3 is increased by at least about 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more as compared to the control. In certain embodiments, an increase in GD3 concentration is observed in a tissue sample such as, for example, the brain. In certain embodiments, an increase in GD3 concentration is observed in the CSF. In certain embodiments, an increase in GD3 concentration is observed in the serum.

[0342] In certain other embodiments, an effective LSD treatment is administered to a subject having an LSD, which results in a decrease in the concentration of at least one GD3 species in a sample from the subject as compared to a control (e.g., the same subject prior to receiving the treatment). In certain embodiments, the concentration of at least one GD3 is decreased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more as compared to the control. In certain embodiments, a decrease in GD3 concentration is observed in a tissue sample such as, for example, the brain. In certain embodiments, a decrease in GD3 concentration is observed in the CSF. In certain embodiments, a decrease in GD3 concentration is observed in the serum.

[0343] The gangliosides GD1a and GD1b are glycosphingolipids. Specific GD1a / b species are described herein (see, e.g., the Examples and Figures).

[0344] In certain embodiments, the concentration of at least one GD1a / b species in a sample from a subject having an LSD is increased as compared to a control (e.g., a healthy control subject not having an LSD). In certain embodiments, GD1a / b is the GD1a / b species described herein, such as, for example, in the Examples or Figures. By way of example, in certain embodiments, GD1a / b is GD1a / b(d36:1) or GD1a / b(d38:1).

[0345] In certain embodiments, the concentration of at least one GD1a / b is increased by at least about 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more as compared to the control. In certain embodiments, an increase in GD1a / b concentration is observed in a tissue sample such as, for example, the brain. In certain embodiments, an increase in GD1a / b concentration is observed in the CSF. In certain embodiments, an increase in GD1a / b concentration is observed in the serum.

[0346] In certain other embodiments, administering an effective LSD treatment to a subject with LSD results in a decrease in the concentration of at least one GD1a / b species in a sample from the subject as compared to a control (e.g., the same subject prior to receiving the treatment). In certain embodiments, the concentration of at least one GD1a / b is decreased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more as compared to the control. In certain embodiments, a decrease in GD1a / b concentration is observed in a tissue sample such as, for example, the brain. In certain embodiments, a decrease in GD1a / b concentration is observed in the CSF. In certain embodiments, a decrease in GD1a / b concentration is observed in the serum.

[0347] Monosialotetrahexosylganglioside 2 (GM2) is a glycosphingolipid. Specific GM2 species are described herein (see, e.g., the Examples and the Figures).

[0348] In certain embodiments, the concentration of at least one GM2 species in a sample from a subject with LSD is increased as compared to a control (e.g., a healthy control subject without LSD). In certain embodiments, the GM2 is a GM2 species described herein, such as, for example, in the Examples or the Figures. By way of example, in certain embodiments, the GM2 species is GM2(d38:1) or GM2(d36:1). In certain embodiments, the concentration of at least one GM2 is increased by at least about 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more as compared to the control. In certain embodiments, an increase in GM2 concentration is observed in a tissue sample such as, for example, the brain. In certain embodiments, an increase in GM2 concentration is observed in the CSF. In certain embodiments, an increase in GM2 concentration is observed in the serum.

[0349] In certain other embodiments, administering an effective LSD treatment to a subject with LSD results in a decrease in the concentration of at least one GM2 species in a sample from the subject as compared to a control (e.g., the same subject prior to receiving the treatment). In certain embodiments, the concentration of at least one GM2 is decreased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more as compared to the control. In certain embodiments, a decrease in GM2 concentration is observed in a tissue sample such as, for example, the brain. In certain embodiments, a decrease in GM2 concentration is observed in the CSF. In certain embodiments, a decrease in GM2 concentration is observed in the serum.

[0350] Similar to GM2, monosialoganglioside 3 (GM3) is also a type of glycosphingolipid. Specific GM3 substances are described herein (see, for example, the Examples and Figures).

[0351] In certain embodiments, the concentration of at least one GM3 substance in a sample from a subject with LSD is increased compared to a control (e.g., a healthy control subject without LSD). In certain embodiments, GM3 is a GM3 substance as described herein, such as in the Examples or Figures. For example, in certain embodiments, the GM3 substance is GM3(d34:1), GM3(d36:1), GM3(d38:1), GM3(d40:1), GM3(d41:1), GM3(d42:2), GM3(d42:1), GM3(d43:0), GM3(d44:1), or GM3(d44:2). In certain embodiments, the GM3 substance is GM3(d34:1), GM3(d36:1), or GM3(d38:1). In certain embodiments, the concentration of at least one GM3 is increased by at least about 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more compared to the control. In certain embodiments, an increase in GM3 concentration is observed in a tissue sample such as the brain. In certain embodiments, an increase in GM3 concentration is observed in the CSF. In certain embodiments, an increase in GM3 concentration is observed in the serum.

[0352] In certain other embodiments, an effective LSD treatment is administered to a subject with LSD, which results in a decrease in the concentration of at least one GM3 in a sample from the subject compared to the control. In certain embodiments, the concentration of at least one GM3 is decreased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more compared to the control. In certain embodiments, a decrease in GM3 concentration is observed in a tissue sample such as the brain. In certain embodiments, a decrease in GM3 concentration is observed in the CSF. In certain embodiments, a decrease in GM3 concentration is observed in the serum.

[0353] Measurement of TREM2, Nf-L, Lipids, and Lipid Combinations

[0354] As used herein, the term "combination of two or more lipids" refers to two or more lipids, wherein at least two lipids are from different classes, and the classes are selected from a) BMP; b) GlcCer; c) GD3 ganglioside; d) GD1a / b ganglioside; and e) GM2 and / or GM3 ganglioside.

[0355] In certain embodiments, a combination of two or more lipids selected from the group consisting of BMP, GlcCer, GD3, GD1a / b, GM2, and GM3 has been evaluated or is being evaluated. In certain embodiments, the combination comprises BMP. In certain embodiments, the combination comprises GlcCer. In certain embodiments, the combination comprises GD3. In certain embodiments, the combination comprises GD1a / b. In certain embodiments, the combination comprises GM2. In certain embodiments, the combination comprises GM3. In certain embodiments, the combination comprises BMP and GlcCer. In certain embodiments, the combination comprises BMP and GD3. In certain embodiments, the combination comprises BMP and GD1a / b. In certain embodiments, the combination comprises BMP and GM2. In certain embodiments, the combination comprises BMP and GM3. In certain embodiments, the combination comprises GlcCer and GD3. In certain embodiments, the combination comprises GlcCer and GD1a / b. In certain embodiments, the combination comprises GlcCer and GM2. In certain embodiments, the combination comprises GlcCer and GM3. In certain embodiments, the combination comprises GD3 and GD1a / b. In certain embodiments, the combination comprises GD3 and GM2. In certain embodiments, the combination comprises GD3 and GM3. In certain embodiments, the combination comprises GD1a / b and GM2. In certain embodiments, the combination comprises GD1a / b and GM3. In certain embodiments, the combination does not consist of GM2 and GM3.

[0356] In certain embodiments, a combination of three or more lipids selected from the group consisting of BMP, GlcCer, GD3, GD1a / b, GM2, and GM3 has been or is being evaluated. In certain embodiments, the combination comprises BMP, GlcCer, and GD3. In certain embodiments, the combination comprises BMP, GlcCer, and GD1a / b. In certain embodiments, the combination comprises BMP, GlcCer, and GM2. In certain embodiments, the combination comprises BMP, GlcCer, and GM3. In certain embodiments, the combination comprises BMP, GD3, and GD1a / b. In certain embodiments, the combination comprises BMP, GD3, and GM2. In certain embodiments, the combination comprises BMP, GD3, and GM3. In certain embodiments, the combination comprises BMP, GD1a / b, and GM2. In certain embodiments, the combination comprises BMP, GD1a / b, and GM3. In certain embodiments, the combination comprises BMP, GM2, and GM3. In certain embodiments, the combination comprises GlcCer, GD3, and GD1a / b. In certain embodiments, the combination comprises GlcCer, GD3, and GM2. In certain embodiments, the combination comprises GlcCer, GD3, and GM3. In certain embodiments, the combination comprises GlcCer, GD1a / b, and GM2. In certain embodiments, the combination comprises GlcCer, GD1a / b, and GM3. In certain embodiments, the combination comprises GlcCer, GM2, and GM3. In certain embodiments, the combination comprises GD3, GD1a / b, and GM2. In certain embodiments, the combination comprises GD3, GD1a / b, and GM3. In certain embodiments, the combination comprises GD3, GM2, and GM3. In certain embodiments, the combination comprises GD1a / b, GM2, and GM3.

[0357] In certain embodiments, a combination of four or more lipids selected from the group consisting of BMP, GlcCer, GD3, GD1a / b, GM2, and GM3 has been evaluated or is being evaluated. In certain embodiments, the combination comprises BMP, GlcCer, GD3, and GD1a / b. In certain embodiments, the combination comprises BMP, GlcCer, GD3, and GM2. In certain embodiments, the combination comprises BMP, GlcCer, GD3, and GM3. In certain embodiments, the combination comprises BMP, GlcCer, GD1a / b, and GM2. In certain embodiments, the combination comprises BMP, GlcCer, GD1a / b, and GM3. In certain embodiments, the combination comprises BMP, GlcCer, GM2, and GM3. In certain embodiments, the combination comprises BMP, GD3, GD1a / b, and GM2. In certain embodiments, the combination comprises BMP, GD3, GD1a / b, and GM3. In certain embodiments, the combination comprises BMP, GD3, GM2, and GM3. In certain embodiments, the combination comprises BMP, GD1a / b, GM2, and GM3. In certain embodiments, the combination comprises GlcCer, GD3, GD1a / b, and GM2. In certain embodiments, the combination comprises GlcCer, GD3, GD1a / b, and GM3. In certain embodiments, the combination comprises GlcCer, GD3, GM2, and GM3. In certain embodiments, the combination comprises GlcCer, GD1a / b, GM2, and GM3. In certain embodiments, the combination comprises GD3, GD1a / b, GM2, and GM3.

[0358] In certain embodiments, a combination of five or more lipids selected from the group consisting of BMP, GlcCer, GD3, GD1a / b, GM2, and GM3 has been evaluated or is being evaluated. In certain embodiments, the combination comprises BMP, GlcCer, GD3, GD1a / b, and GM2. In certain embodiments, the combination comprises BMP, GlcCer, GD3, GD1a / b, and GM3. In certain embodiments, the combination comprises BMP, GD3, GD1a / b, GM2, and GM3. In certain embodiments, the combination comprises BMP, GlcCer, GD3, GM2, and GM3. In certain embodiments, the combination comprises BMP, GlcCer, GD1a / b, GM2, and GM3. In certain embodiments, the combination comprises GlcCer, GD3, GD1a / b, GM2, and GM3.

[0359] In certain embodiments, a combination of BMP, GlcCer, GD3, GD1a / b, GM2, and GM3 has been evaluated or is being evaluated.

[0360] In certain embodiments, sTREM2 has been or is being evaluated. In certain embodiments, sTREM2 and one or more lipids have been or are being evaluated. In certain embodiments, the lipid is BMP. In certain embodiments, the lipid is GlcCer. In certain embodiments, the lipid is GD3. In certain embodiments, the lipid is GD1a / b. In certain embodiments, the lipid is GM2. In certain embodiments, the lipid is GM3.

[0361] In certain embodiments, sTREM2 and a combination of two or more lipids selected from the group consisting of BMP, GlcCer, GD3, GD1a / b, GM2, and GM3 have been or are being evaluated. In certain embodiments, the combination of two or more lipids is the combination described herein.

[0362] In certain embodiments, sTREM2 and a combination of three or more lipids selected from the group consisting of BMP, GlcCer, GD3, GD1a / b, GM2, and GM3 have been or are being evaluated. In certain embodiments, the combination of three or more lipids is the combination described herein.

[0363] In certain embodiments, sTREM2 and a combination of four or more lipids selected from the group consisting of BMP, GlcCer, GD3, GD1a / b, GM2, and GM3 have been or are being evaluated. In certain embodiments, the combination of four or more lipids is the combination described herein.

[0364] In certain embodiments, sTREM2 and a combination of five or more lipids selected from the group consisting of BMP, GlcCer, GD3, GD1a / b, GM2, and GM3 have been or are being evaluated. In certain embodiments, the combination of five or more lipids is the combination described herein.

[0365] In certain embodiments, sTREM2, BMP, GlcCer, GD3, GD1a / b, GM2, and GM3 have been or are being evaluated.

[0366] In certain embodiments, Nf-L has been or is being evaluated.

[0367] In certain embodiments, Nf-L and sTREM2 have been or are being evaluated.

[0368] In certain embodiments, Nf-L and one or more lipids have been or are being evaluated. In certain embodiments, the lipid is BMP. In certain embodiments, the lipid is GlcCer. In certain embodiments, the lipid is GD3. In certain embodiments, the lipid is GD1a / b. In certain embodiments, the lipid is GM2. In certain embodiments, the lipid is GM3.

[0369] In certain embodiments, Nf-L and a combination of two or more lipids selected from the group consisting of BMP, GlcCer, GD3, GD1a / b, GM2, and GM3 have been or are being evaluated. In certain embodiments, the combination of two or more lipids is the combination described herein.

[0370] In certain embodiments, Nf-L and a combination of three or more lipids selected from the group consisting of BMP, GlcCer, GD3, GD1a / b, GM2, and GM3 have been or are being evaluated. In certain embodiments, the combination of three or more lipids is the combination described herein.

[0371] In certain embodiments, Nf-L and a combination of four or more lipids selected from the group consisting of BMP, GlcCer, GD3, GD1a / b, GM2, and GM3 have been or are being evaluated. In certain embodiments, the combination of four or more lipids is the combination described herein.

[0372] In certain embodiments, Nf-L and a combination of five or more lipids selected from the group consisting of BMP, GlcCer, GD3, GD1a / b, GM2, and GM3 have been or are being evaluated. In certain embodiments, the combination of five or more lipids is the combination described herein.

[0373] In certain embodiments, Nf-L, BMP, GlcCer, GD3, GD1a / b, GM2, and GM3 have been or are being evaluated.

[0374] In certain embodiments, sTREM2, Nf-L, and one or more lipids have been or are being evaluated. In certain embodiments, sTREM2, Nf-L, BMP, GlcCer, GD3, GD1a / b, GM2, and GM3 have been or are being evaluated.

[0375] Thus, samples obtained from a subject having or suspected of having an LSD can be evaluated for the accumulation of sTREM2, Nf-L, and / or one or more lipids selected from BMP, GlcCer, GD3, GD1a / b, GM2, and GM3. Specifically, the concentration of sTREM2 protein, Nf-L, and / or one or more lipids in a sample obtained from a subject can be measured using assays known in the art or described herein (e.g., mass spectrometry).

[0376] In certain embodiments, the concentration of the protein and / or lipid is compared to the concentration of the corresponding protein and / or lipid in a sample from a control subject (e.g., a healthy subject without LSD).

[0377] In some embodiments, the amount of each selected lipid / protein in a sample from a subject is compared to a control value determined for a healthy control or healthy control population (i.e., not having LSD). In some embodiments, if the amount of each selected lipid / protein in a sample from a subject is increased compared to the control value, then the subject is identified as a candidate for treatment or treatment adjustment. In some embodiments, if the amount of each selected lipid / protein in a sample from a subject is increased by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more compared to the control value, then the subject is identified as a candidate for treatment or treatment adjustment. In some embodiments, if the amount of each selected lipid / protein in a sample from a subject is increased by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more compared to the control value, then the subject is identified as a candidate for treatment or treatment adjustment. In some embodiments, the healthy control value for each selected lipid / protein is determined by assessing the level of each lipid / protein in a subject or group of subjects known not to have LSD (e.g., 10, 20, 50, 100, 200, 500, 1000 subjects or more).

[0378] In some embodiments, the amount of each selected lipid / protein in a sample from a subject is compared to a control value determined for a disease control or disease control population (i.e., having LSD). In some embodiments, the disease control value for each selected lipid / protein is determined by assessing the level of the selected lipid / protein in a subject or group of subjects known to have LSD (e.g., 10, 20, 50, 100, 200, 500, 1000 subjects or more).

[0379] In some embodiments, a subject is identified as a candidate for treatment or treatment adjustment (e.g., increased dose or frequency) if the amount of each selected lipid / protein in a sample from the subject is at least as high as the amount of each selected lipid / protein in a disease control or disease control population. In some embodiments, a subject is identified as a candidate for treatment or treatment adjustment (e.g., increased dose or frequency) if the amount of each selected lipid / protein in a sample from the subject is comparable to the amount of each selected lipid / protein in a disease control or disease control population (e.g., within 20%, 10%, 5%, 4%, 3%, 2%, or 1%).

[0380] In some embodiments, a subject with an LSD is a subject who has been administered a treatment for the LSD. The levels in the subject after treatment are compared to the levels in the same subject before administration of the treatment (e.g., before the first administration). The effectiveness of the treatment can be determined by a change (e.g., a decrease) in the amount of the selected lipid / protein.

[0381] In certain other embodiments, a subject is identified as a candidate for treatment adjustment (e.g., decreased dose or frequency) if the amount of each selected lipid / protein in a sample from the subject is less than the amount of each selected lipid / protein in a disease control or disease control population.

[0382] In some embodiments, a population of subjects is matched to a test subject based on one or more patient characteristics, such as age, gender, race, or other criteria. In some embodiments, control values are established using samples of the same type as those used to assess lipid / protein levels in the test subject from the population of subjects (e.g., samples comprising blood or PBMC).

[0383] Lysosomal storage disease

[0384] As described herein, certain biomarkers associated with LSDs have been identified. LSDs are inherited metabolic disorders characterized by the accumulation of undigested or partially digested macromolecules, ultimately leading to cellular dysfunction and clinical abnormalities. Traditionally, LSDs have been defined as lysosomal function deficiencies generally classified by the accumulating substrate and include mucopolysaccharidoses. More recently, the classification of these disorders has been expanded to include other protein deficiencies or defects that cause macromolecule accumulation, such as proteins required for normal post-translational modification of lysosomal enzymes, or proteins important for proper lysosomal trafficking.

[0385] In certain embodiments, the LSD is an MPS disorder (e.g., Hunter syndrome).

[0386] Therapeutic agent

[0387] Certain methods described herein include administering a lysosomal storage disorder treatment to a subject.

[0388] As used herein, "lysosomal storage disorder treatment" can be any therapeutic agent or therapy capable of reducing one or more symptoms associated with an LSD (e.g., neurological symptoms). Certain lysosomal storage disorder treatments are known. By way of example, such treatments include, for example, hematopoietic stem cell transplantation (HSCT), enzyme replacement therapy (ERT), substrate reduction therapy, chaperone therapy, and gene therapy (e.g., in vivo or ex vivo).

[0389] In certain embodiments, the LSD treatment comprises ERT. In certain embodiments, the ERT can be a therapy designed to treat one or more neurological symptoms. As described below, certain ERT LSD therapies can target the brain using an enzyme transport vehicle (ETV). By way of example, the following discussion includes certain fusion proteins of ERT enzymes that can be used in the methods described herein and are described in WO 2019 / 070577 (incorporated herein by reference in its entirety for all purposes).

[0390] Certain fusion proteins comprising an ERT enzyme

[0391] Certain embodiments of fusion proteins are described below, which fusion proteins comprise an enzyme replacement therapy (ERT) enzyme linked to an Fc polypeptide; these fusion proteins can be used as an LSD treatment in certain methods described herein. In some cases, the protein comprises a dimeric Fc polypeptide, wherein one of the Fc polypeptide monomers is linked to the ERT enzyme. The Fc polypeptide can increase the enzyme half-life and, in some cases, can be modified to confer additional functionality to the protein. Fusion proteins that facilitate delivery of the ERT enzyme across the blood-brain barrier (BBB) are also described herein. These proteins comprise an Fc polypeptide and a modified Fc polypeptide that form a dimer, and an ERT enzyme linked to the Fc region and / or the modified Fc region. The modified Fc region can specifically bind to a BBB receptor, such as the transferrin receptor (TfR). In some embodiments, the ERT enzyme is iduronate-2-sulfatase (IDS), or a catalytically active variant or fragment of wild-type IDS, such as wild-type human IDS. Certain embodiments of these fusion proteins can be referred to herein as enzyme transport vehicles (ETV) in association with a specific enzyme, e.g., ETV:IDS.

[0392] ERT enzyme

[0393] In some aspects, the fusion proteins described herein comprise: (i) an Fc polypeptide that may contain modifications (such as one or more modifications that promote heterodimerization) or may be a wild-type Fc polypeptide, and an ERT enzyme; and (ii) an Fc polypeptide that may contain modifications (such as one or more modifications that promote heterodimerization) or may be a wild-type Fc polypeptide, and optionally an ERT enzyme. In some embodiments, one or both Fc polypeptides may contain modifications that cause binding to a blood-brain barrier (BBB) receptor such as TfR. The ERT enzyme can be any enzyme that is deficient in an LSD. The ERT enzyme incorporated into the fusion protein is catalytically active, i.e., retains the enzymatic activity that is deficient in the LSD. In some embodiments, the ERT enzyme is IDS, which is deficient in Hunter syndrome.

[0394] In some embodiments, the fusion protein comprising an ERT enzyme and optionally a modified Fc polypeptide that binds to a BBB receptor (such as an Fc polypeptide that binds TfR) comprises a catalytically active fragment or variant of wild-type IDS. In some embodiments, the IDS enzyme is a variant or catalytically active fragment of an IDS protein comprising the amino acid sequence of any one of SEQ ID NOs: 91, 92, 112, 192, and 196. In some embodiments, the catalytically active variant or fragment of the IDS enzyme has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more of the activity of the wild-type IDS enzyme.

[0395] In some embodiments, the ERT enzyme (such as IDS) or its catalytically active variant or fragment present in the fusion proteins described herein retains at least 25% of its activity compared to its activity when it is not conjugated to an Fc polypeptide or an Fc polypeptide that binds TfR. In some embodiments, the ERT enzyme or its catalytically active variant or fragment retains at least 10%, or at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of its activity compared to its activity when it is not conjugated to an Fc polypeptide or an Fc polypeptide that binds TfR. In some embodiments, the ERT enzyme or its catalytically active variant or fragment retains at least 80%, 85%, 90% or 95% of its activity compared to its activity when it is not conjugated to an Fc polypeptide or an Fc polypeptide that binds TfR. In some embodiments, fusion to an Fc polypeptide does not reduce the activity of an ERT enzyme such as IDS or its catalytically active variant or fragment. In some embodiments, fusion to an Fc polypeptide that binds TfR does not reduce the activity of the ERT enzyme.

[0396] I. Modifications of Fc Polypeptides for Binding to Blood-Brain Barrier (BBB) Receptors

[0397] In some aspects, the fusion protein is capable of transporting across the blood-brain barrier (BBB). This protein comprises a modified Fc polypeptide that binds to a BBB receptor. The BBB receptor is expressed on BBB endothelium and other cell and tissue types. In some embodiments, the BBB receptor is the transferrin receptor (TfR).

[0398] In this document, EU index numbers are used to number the various Fc modifications, including the amino acid residues represented in the modifications introduced in the modified Fc polypeptides that bind to BBB receptors such as TfR. Any Fc polypeptide, such as an IgG1, IgG2, IgG3, or IgG4 Fc polypeptide, may have modifications, such as amino acid substitutions, at one or more positions as described herein.

[0399] The modified (e.g., enhanced heterodimerization and / or BBB receptor binding) Fc polypeptide present in the fusion proteins described herein may have at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with the native Fc region sequence or a fragment thereof, such as at least 50 amino acids or at least 100 amino acids or a longer fragment. In some embodiments, the native Fc amino acid sequence is the Fc region sequence of SEQ ID NO:1. In some embodiments, the modified Fc polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with amino acids 1-110 of SEQ ID NO:1, or with amino acids 111-217 of SEQ ID NO:1, or a fragment thereof, such as at least 50 amino acids or at least 100 amino acids or a longer fragment.

[0400] In some embodiments, the modified (e.g., enhanced heterodimerization and / or BBB receptor binding) Fc polypeptide comprises at least 50 amino acids corresponding to the native Fc region amino acid sequence, or at least 60, 65, 70, 75, 80, 85, 90, or 95 or more or at least 100 amino acids or more. In some embodiments, the modified Fc polypeptide comprises at least 25 contiguous amino acids corresponding to the native Fc region amino acid sequence of, for example, SEQ ID NO:1, or at least 30, 35, 40, or 45 contiguous amino acids, or 50 contiguous amino acids, or at least 60, 65, 70, 75, 80, 85, 90, or 95 or more contiguous amino acids, or 100 or more contiguous amino acids.

[0401] In some embodiments, the domain modified for BBB receptor binding activity is a human Ig CH3 domain, such as the IgG1 CH3 domain. The CH3 domain can be of any IgG subtype, namely IgG1, IgG2, IgG3 or IgG4. In the context of an IgG1 antibody, the CH3 domain refers to the amino acid segment from approximately position 341 to approximately position 447 as numbered according to the EU numbering scheme.

[0402] In some embodiments, the modified (e.g., BBB receptor binding) Fc polypeptide present in the fusion proteins described herein contains at least one, two or three substitutions at amino acid positions 384, 386, 387, 388, 389, 390, 413, 416 and 421 according to the EU numbering scheme; and in some embodiments, at least four, five, six, seven, eight or nine substitutions.

[0403] FcRn binding site

[0404] In certain aspects, the modified (e.g., BBB receptor binding) Fc polypeptide or the Fc polypeptide that does not specifically bind to the BBB receptor present in the fusion proteins described herein can also contain an FcRn binding site. In some embodiments, the FcRn binding site is within the Fc polypeptide or a fragment thereof.

[0405] In some embodiments, the FcRn binding site comprises a native FcRn binding site. In some embodiments, the FcRn binding site does not contain amino acid changes relative to the amino acid sequence of the native FcRn binding site. In some embodiments, the native FcRn binding site is an IgG binding site, such as a human IgG binding site. In some embodiments, the FcRn binding site contains a modification that alters FcRn binding.

[0406] In some embodiments, the FcRn binding site has one or more mutations, such as substituted amino acid residues, wherein the mutation increases the serum half-life or does not substantially reduce the serum half-life (i.e., the serum half-life is reduced by at most 25% compared to the corresponding modified Fc polypeptide having a wild-type residue at the mutated position when measured under the same conditions). In some embodiments, the FcRn binding site has one or more amino acid residues substituted at positions 250 - 256, 307, 380, 428 and 433 - 436 according to the EU numbering scheme.

[0407] In some embodiments, one or more residues at or near the FcRn binding site are mutated relative to the native human IgG sequence to extend the serum half-life of the modified polypeptide. In some embodiments, the mutation is introduced into one, two, or three of positions 252, 254, and 256. In some embodiments, the mutations are M252Y, S254T, and T256E. In some embodiments, the modified Fc polypeptide also comprises the mutations M252Y, S254T, and T256E. In some embodiments, the modified Fc polypeptide comprises substitutions at one, two, or all three of positions T307, E380, and N434 according to the EU numbering scheme. In some embodiments, the mutations are T307Q and N434A. In some embodiments, the modified Fc polypeptide comprises the mutations T307A, E380A, and N434A. In some embodiments, the modified Fc polypeptide comprises substitutions at positions T250 and M428 according to the EU numbering scheme. In some embodiments, the modified Fc polypeptide comprises the mutation T250Q and / or M428L. In some embodiments, the modified Fc polypeptide comprises substitutions at positions M428 and N434 according to the EU numbering scheme. In some embodiments, the modified Fc polypeptide comprises the mutations M428L and N434S. In some embodiments, the modified Fc polypeptide comprises the N434S or N434A mutation.

[0408] II. Fc Polypeptides That Bind Transferrin Receptor

[0409] This section describes the generation of the modified Fc polypeptides described herein that bind to the transferrin receptor (TfR) and are capable of transporting across the blood-brain barrier (BBB).

[0410] Fc Polypeptides That Bind TfR and Contain Mutations in the CH3 Domain

[0411] In some embodiments, the modified Fc polypeptide that specifically binds to TfR contains substitutions in the CH3 domain. In some embodiments, the modified Fc polypeptide comprises a human Ig CH3 domain modified for TfR binding activity, such as an IgG CH3 domain. The CH3 domain may belong to any IgG subtype, i.e., IgG1, IgG2, IgG3, or IgG4. In the context of an IgG antibody, the CH3 domain refers to the amino acid segment from approximately position 341 to approximately position 447 as numbered according to the EU numbering scheme.

[0412] In some embodiments, a modified Fc polypeptide that specifically binds to TfR binds to the apical domain of TfR and can bind to TfR without blocking or otherwise inhibiting the binding of transferrin to TfR. In some embodiments, the binding of transferrin to TfR is substantially not inhibited. In some embodiments, the binding of transferrin to TfR is inhibited by less than about 50% (e.g., less than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%). In some embodiments, the binding of transferrin to TfR is inhibited by less than about 20% (e.g., less than about 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%).

[0413] In some embodiments, a modified Fc polypeptide that specifically binds to TfR comprises at least two, three, four, five, six, seven, eight, or nine substitutions at amino acid positions 384, 386, 387, 388, 389, 390, 413, 416, and 421 according to the EU numbering scheme. Exemplary substitutions that can be introduced at these positions are shown in Tables 4 and 5. In some embodiments, the amino acid at position 388 and / or 421 is an aromatic amino acid, such as Trp, Phe, or Tyr. In some embodiments, the amino acid at position 388 is Trp. In some embodiments, the aromatic amino acid at position 421 is Trp or Phe.

[0414] In some embodiments, at least one of the following positions is substituted: Leu, Tyr, Met, or Val at position 384; Leu, Thr, His, or Pro at position 386; Val, Pro, or an acidic amino acid at position 387; an aromatic amino acid, such as Trp, at position 388; Val, Ser, or Ala at position 389; an acidic amino acid, Ala, Ser, Leu, Thr, or Pro at position 413; Thr or an acidic amino acid at position 416; or Trp, Tyr, His, or Phe at position 421. In some embodiments, the modified Fc polypeptide can comprise conservative substitutions, such as amino acids within the same charge grouping, hydrophobic grouping, side chain cyclic structure grouping (e.g., aromatic amino acids), or size grouping and / or polar or nonpolar grouping of the designated amino acid at one or more of the positions in the set. Thus, for example, Ile can be present at position 384, 386, and / or position 413. In some embodiments, the acidic amino acid at one, two, or each of positions 387, 413, and 416 is Glu. In other embodiments, the acidic amino acid at one, two, or each of positions 387, 413, and 416 is Asp. In some embodiments, two, three, four, five, six, seven, or all eight of positions 384, 386, 387, 388, 389, 413, 416, and 421 have amino acid substitutions as described in this paragraph.

[0415] In some embodiments, the Fc polypeptide modified as described in the previous two paragraphs comprises a native Asn at position 390. In some embodiments, the modified Fc polypeptide comprises Gly, His, Gln, Leu, Lys, Val, Phe, Ser, Ala, or Asp at position 390. In some embodiments, the modified Fc polypeptide further comprises one, two, three, or four substitutions at positions 380, 391, 392, and 415 according to the EU numbering scheme. In some embodiments, Trp, Tyr, Leu, or Gln can be present at position 380. In some embodiments, Ser, Thr, Gln, or Phe can be present at position 391. In some embodiments, Gln, Phe, or His can be present at position 392. In some embodiments, Glu can be present at position 415.

[0416] In certain embodiments, a modified Fc polypeptide comprises two, three, four, five, six, seven, eight, nine, ten, or eleven positions selected from the following: Trp, Leu, or Glu at position 380; Tyr or Phe at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ser, Ala, Val, or Asn at position 389; Ser or Asn at position 390; Thr or Ser at position 413; Glu or Ser at position 415; Glu at position 416; and / or Phe at position 421. In some embodiments, a modified Fc polypeptide comprises all eleven of the following positions: Trp, Leu, or Glu at position 380; Tyr or Phe at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ser, Ala, Val, or Asn at position 389; Ser or Asn at position 390; Thr or Ser at position 413; Glu or Ser at position 415; Glu at position 416; and / or Phe at position 421.

[0417] In certain embodiments, a modified Fc polypeptide comprises Leu or Met at position 384; Leu, His, or Pro at position 386; Val at position 387; Trp at position 388; Val or Ala at position 389; Pro at position 413; Thr at position 416; and / or Trp at position 421. In some embodiments, the modified Fc polypeptide further comprises Ser, Thr, Gln, or Phe at position 391. In some embodiments, the modified Fc polypeptide further comprises Trp, Tyr, Leu, or Gln at position 380 and / or Gln, Phe, or His at position 392. In some embodiments, Trp is present at position 380 and / or Gln is present at position 392. In some embodiments, the modified Fc polypeptide does not have Trp at position 380.

[0418] In other embodiments, the modified Fc polypeptide comprises Tyr at position 384; Thr at position 386; Glu or Val and position 387; Trp at position 388; Ser at position 389; Ser or Thr at position 413; Glu at position 416; and / or Phe at position 421. In some embodiments, the modified Fc polypeptide comprises the native Asn at position 390. In certain embodiments, the modified Fc polypeptide further comprises Trp, Tyr, Leu or Gln at position 380; and / or Glu at position 415. In some embodiments, the modified Fc polypeptide further comprises Trp at position 380 and / or Glu at position 415.

[0419] In additional embodiments, the modified Fc polypeptide further comprises one, two or three substitutions at positions comprising 414, 424 and 426 according to the EU numbering scheme. In some embodiments, position 414 is Lys, Arg, Gly or Pro; position 424 is Ser, Thr, Glu or Lys; and / or position 426 is Ser, Trp or Gly.

[0420] In some embodiments, the modified Fc polypeptide comprises one or more of the following substitutions: Trp at position 380 according to the EU numbering scheme; Thr at position 386; Trp at position 388; Val at position 389; Thr or Ser at position 413; Glu at position 415; and / or Phe at position 421.

[0421] In some embodiments, the modified Fc polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with amino acids 111-217 of any one of SEQ ID NOs: 4-90, 95-98, and 103-106 (such as SEQ ID NOs: 34-38, 58, and 60-90). In some embodiments, the modified Fc polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with any one of SEQ ID NOs: 4-90, 95-98, and 103-106 (such as SEQ ID NOs: 34-38, 58, and 60-90). In some embodiments, the modified Fc polypeptide comprises amino acids at EU index positions 384-390 and / or 413-421 of any one of SEQ ID NOs: 4-90, 95-98, and 103-106 (such as SEQ ID NOs: 34-38, 58, and 60-90). In some embodiments, the modified Fc polypeptide comprises amino acids at EU index positions 380-390 and / or 413-421 of any one of SEQ ID NOs: 4-90, 95-98, and 103-106 (such as SEQ ID NOs: 34-38, 58, and 60-90). In some embodiments, the modified Fc polypeptide comprises amino acids at EU index positions 380-392 and / or 413-426 of any one of SEQ ID NOs: 4-90, 95-98, and 103-106 (such as SEQ ID NOs: 34-38, 58, and 60-90).

[0422] In some embodiments, the modified Fc polypeptide has at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with any one of SEQ ID NOs: 4-90, 95-98, and 103-106 (such as SEQ ID NOs: 34-38, 58, and 60-90), and further comprises at least five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen of the following positions according to EU index numbers: Trp, Tyr, Leu, Gln, or Glu at position 380; Leu, Tyr, Met, or Val at position 384; Leu, Thr, His, or Pro at position 386; Val, Pro, or acidic amino acid at position 387; aromatic amino acid, such as Trp, at position 388; Val, Ser, or Ala at position 389; Ser or Asn at position 390; Ser, Thr, Gln, or Phe at position 391; Gln, Phe, or His at position 392; acidic amino acid, Ala, Ser, Leu, Thr, or Pro at position 413; Lys, Arg, Gly, or Pro at position 414; Glu or Ser at position 415; Thr or acidic amino acid at position 416; Trp, Tyr, His, or Phe at position 421; Ser, Thr, Glu, or Lys at position 424; and Ser, Trp, or Gly at position 426.

[0423] In some embodiments, the modified Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 34-38, 58, and 60-90. In other embodiments, the modified Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 34-38, 58, and 60-90, but one, two, or three amino acids are substituted.

[0424] In some embodiments, the modified Fc polypeptide comprises additional mutations, such as those described below, including (but not limited to) the staphylococcal nuclease (SN) mutation (e.g., T366W numbered with reference to the EU numbering), the SNAP25 mutation (e.g., T366S, L368A, and Y407V numbered with reference to the EU numbering), mutations that modulate effector function (e.g., L234A, L235A, and / or P329G (e.g., L234A and L235A) numbered with reference to the EU numbering), and / or mutations that increase serum stability or serum half-life (e.g., (i) M252Y, S254T, and T256E numbered with reference to the EU numbering, or (ii) N434S numbered with reference to the EU numbering, with or without M428L). By way of illustration, SEQ ID NOs: 118 - 191 provide non-limiting examples of modified Fc polypeptides having mutations in the CH3 domain that include one or more of these additional mutations (e.g., Clone CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, and CH3C.35.23).

[0425] In some embodiments, the modified Fc polypeptide comprises the staphylococcal nuclease (SN) mutation (e.g., T366W numbered with reference to the EU numbering) and has at least 85%, at least 90%, or at least 95% identity to the sequence of any one of SEQ ID NOs: 118, 130, 142, 154, 166, and 178. In some embodiments, the modified Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 118, 130, 142, 154, 166, and 178.

[0426] In some embodiments, the modified Fc polypeptide comprises the staphylococcal nuclease (SN) mutation (e.g., T366W numbered with reference to the EU numbering) and mutations that modulate effector function (e.g., L234A, L235A, and / or P329G (e.g., L234A and L235A) numbered with reference to the EU numbering), and has at least 85%, at least 90%, or at least 95% identity to the sequence of any one of SEQ ID NOs: 119, 120, 131, 132, 143, 144, 155, 156, 167, 168, 179, 180, 190, and 191. In some embodiments, the modified Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 119, 120, 131, 132, 143, 144, 155, 156, 167, 168, 179, and 180.

[0427] In some embodiments, the modified Fc polypeptide comprises a staphylococcal nuclease mutation (e.g., T366W numbered with reference to the EU numbering) and a mutation that increases serum stability or serum half-life (e.g., (i) M252Y, S254T, and T256E numbered with reference to the EU numbering, or (ii) N434S numbered with reference to the EU numbering, with or without M428L), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 121, 133, 145, 157, 169, and 181. In some embodiments, the modified Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 121, 133, 145, 157, 169, and 181.

[0428] In some embodiments, the modified Fc polypeptide comprises a staphylococcal nuclease mutation (e.g., T366W numbered with reference to the EU numbering), a mutation that modulates effector function (e.g., L234A, L235A, and / or P329G (e.g., L234A and L235A) numbered with reference to the EU numbering), and a mutation that increases serum stability or serum half-life (e.g., (i) M252Y, S254T, and T256E numbered with reference to the EU numbering, or (ii) N434S numbered with reference to the EU numbering, with or without M428L), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 122, 123, 134, 135, 146, 147, 158, 159, 170, 171, 182, and 183. In some embodiments, the modified Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 122, 123, 134, 135, 146, 147, 158, 159, 170, 171, 182, and 183.

[0429] In some embodiments, the modified Fc polypeptide comprises a papain mutation (e.g., T366S, L368A, and Y407V numbered with reference to the EU numbering) and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 124, 136, 148, 160, 172, and 184. In some embodiments, the modified Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 124, 136, 148, 160, 172, and 184.

[0430] In some embodiments, the modified Fc polypeptide comprises a CH2 mutation (e.g., T366S, L368A, and Y407V numbered with reference to EU numbering) and a mutation that modulates effector function (e.g., L234A, L235A, and / or P329G (e.g., L234A and L235A) numbered with reference to EU numbering), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 125, 126, 137, 138, 149, 150, 161, 162, 173, 174, 185, and 186. In some embodiments, the modified Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 125, 126, 137, 138, 149, 150, 161, 162, 173, 174, 185, and 186.

[0431] In some embodiments, the modified Fc polypeptide comprises a CH2 mutation (e.g., T366S, L368A, and Y407V numbered with reference to EU numbering) and a mutation that increases serum stability or serum half-life (e.g., (i) M252Y, S254T, and T256E numbered with reference to EU numbering, or (ii) N434S numbered with reference to EU numbering, with or without M428L), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 127, 139, 151, 163, 175, and 187. In some embodiments, the modified Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 127, 139, 151, 163, 175, and 187.

[0432] In some embodiments, the modified Fc polypeptide comprises mutations in the CH2 domain (e.g., T366S, L368A, and Y407V numbered with reference to EU numbering), mutations that modulate effector function (e.g., L234A, L235A, and / or P329G (e.g., L234A and L235A) numbered with reference to EU numbering), and mutations that increase serum stability or serum half-life (e.g., (i) M252Y, S254T, and T256E numbered with reference to EU numbering, or (ii) N434S numbered with reference to EU numbering, with or without M428L), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 128, 129, 140, 141, 152, 153, 164, 165, 176, 177, 188, and 189. In some embodiments, the modified Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 128, 129, 140, 141, 152, 153, 164, 165, 176, 177, 188, and 189.

[0433] In some embodiments, the modified Fc polypeptide that specifically binds to TfR comprises at least two, three, four, five, six, seven, or eight substitutions at positions 345, 346, 347, 349, 437, 438, 439, and 440 according to the EU numbering scheme. In some embodiments, the modified Fc polypeptide comprises Gly at position 437; Phe at position 438; and / or Asp at position 440. In some embodiments, Glu is present at position 440. In certain embodiments, the modified Fc polypeptide comprises at least one substitution at the following positions: Phe or Ile at position 345; Asp, Glu, Gly, Ala, or Lys at position 346; Tyr, Met, Leu, Ile, or Asp at position 347; Thr or Ala at position 349; Gly at position 437; Phe at position 438; His, Tyr, Ser, or Phe at position 439; or Asp at position 440. In some embodiments, as described in this paragraph, two, three, four, five, six, seven, or all eight of positions 345, 346, 347, 349, 437, 438, 439, and 440 have substitutions. In some embodiments, the modified Fc polypeptide may comprise conservative substitutions, such as amino acids in the same charge grouping, hydrophobic grouping, side-chain cyclic structure grouping (e.g., aromatic amino acids), or size grouping and / or polar or nonpolar grouping of the designated amino acid at one or more positions in the set.

[0434] III. Additional Fc Polypeptide Mutations

[0435] In some aspects, the fusion proteins described herein comprise two Fc polypeptides, which may each comprise independently selected modifications, or may be wild-type Fc polypeptides, such as human IgG1 Fc polypeptides. In some embodiments, one or both of the Fc polypeptides contain one or more modifications that confer binding to a blood-brain barrier (BBB) receptor, such as the transferrin receptor (TfR). Non-limiting examples of other mutations that may be introduced into one or both of the Fc polypeptides include, for example, mutations that increase serum stability or serum half-life, modulate effector function, affect glycosylation, reduce immunogenicity in humans, and / or provide for knobs-into-holes heterodimerization of the Fc polypeptides.

[0436] In some embodiments, the Fc polypeptides present in the fusion protein independently have at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the corresponding wild-type Fc polypeptide (such as a human IgG1, IgG2, IgG3 or IgG4 Fc polypeptide).

[0437] In some embodiments, the Fc polypeptides present in the fusion protein comprise knobs and holes mutations to promote heterodimer formation and impede homodimer formation. Generally, the modification introduces a protrusion ("knob") at the interface of the first polypeptide and a corresponding cavity ("hole") in the interface of the second polypeptide such that the protrusion can fit into the cavity to promote heterodimer formation and thus impede homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain, such as tyrosine or tryptophan. A compensatory cavity of the same or similar size as the protrusion is created in the interface of the second polypeptide by replacing a large amino acid side chain with a smaller amino acid side chain, such as alanine or threonine. In some embodiments, such additional mutations are in positions in the Fc polypeptide that have no side effects on the binding of the polypeptide to a BBB receptor such as TfR.

[0438] In a schematic embodiment of the chimeric method for dimerization, position 366 (numbered according to the EU numbering scheme) of one of the Fc polypeptides present in the fusion protein contains tryptophan instead of the native threonine. Another Fc polypeptide in the dimer has valine at position 407 (numbered according to the EU numbering scheme) instead of the native tyrosine. Another Fc polypeptide may also contain substitutions where the native threonine at position 366 (numbered according to the EU numbering scheme) is replaced by serine and the native leucine at position 368 (numbered according to the EU numbering scheme) is replaced by alanine. Thus, one Fc polypeptide of the fusion protein described herein has a T366W chimeric mutation and another Fc polypeptide has a Y407V mutation, which is typically accompanied by T366S and L368A socket mutations.

[0439] In some embodiments, modifications that enhance serum half-life can be introduced. For example, in some embodiments, one or both Fc polypeptides present in the fusion protein described herein can contain tyrosine at position 252, threonine at position 254, and glutamic acid at position 256, as numbered according to the EU numbering scheme. Thus, one or both Fc polypeptides can have M252Y, S254T, and T256E substitutions. Alternatively, one or both Fc polypeptides can have M428L and N434S substitutions, as numbered according to the EU numbering scheme. Alternatively, one or both Fc polypeptides can have N434S or N434A substitutions.

[0440] In some embodiments, one or both Fc polypeptides present in the fusion protein described herein can be engineered to contain modifications that reduce effector function, i.e., the ability to induce certain biological functions after binding to Fc receptors expressed on effector cells that mediate effector functions. Examples of antibody effector functions include (but are not limited to) C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Effector functions can vary with antibody class. For example, native human IgG1 and IgG3 antibodies can elicit ADCC and CDC activities after binding to appropriate Fc receptors present on immune system cells, and native human IgG1, IgG2, IgG3, and IgG4 can elicit ADCP function after binding to appropriate Fc receptors present on immune cells.

[0441] In some embodiments, one or both Fc polypeptides present in the fusion protein described herein can also be engineered to contain other modifications for heterodimerization, such as electrostatic engineering of the contacting residues within the CH3-CH3 interface, which are native charged or hydrophobic patch modifications.

[0442] In some embodiments, one or both of the Fc polypeptides present in the fusion proteins described herein may include additional modifications that modulate effector function.

[0443] In some embodiments, one or both of the Fc polypeptides present in the fusion proteins described herein may contain modifications that reduce or eliminate effector function. Exemplary Fc polypeptide mutations that reduce effector function include (but are not limited to) substitutions at positions 234 and 235 in the CH2 domain, for example, according to the EU numbering scheme. By way of example, in some embodiments, one or both of the Fc polypeptides may contain alanine residues at positions 234 and 235. Accordingly, one or both of the Fc polypeptides may have L234A and L235A (LALA) substitutions.

[0444] Additional Fc polypeptide mutations that modulate effector function include (but are not limited to) the following: position 329 may have a mutation where proline is substituted with glycine or arginine or an amino acid residue large enough to disrupt the Fc / Fcγ receptor interface formed between proline 329 of Fc and the tryptophan residues Trp 87 and Trp 110 of FcγRIII. Additional exemplary substitutions include S228P, E233P, L235E, N297A, N297D, and P331S according to the EU numbering scheme. Multiple substitutions may also be present, such as L234A and L235A in the human IgG1 Fc region according to the EU numbering scheme; L234A, L235A, and P329G in the human IgG1 Fc region; S228P and L235E in the human IgG4 Fc region; L234A and G237A in the human IgG1 Fc region; L234A, L235A, and G237A in the human IgG1 Fc region; V234A and G237A in the human IgG2 Fc region; L235A, G237A, and E318A in the human IgG4 Fc region; and S228P and L236E in the human IgG4 Fc region. In some embodiments, one or both of the Fc polypeptides may have one or more amino acid substitutions that modulate ADCC, such as substitutions at positions 298, 333, and / or 334 according to the EU numbering scheme.

[0445] Exemplary Fc polypeptides containing additional mutations

[0446] By way of non-limiting example, one or both of the Fc polypeptides present in the fusion proteins described herein can contain additional mutations, including a knuckle mutation (e.g., T366W as numbered according to the EU numbering scheme), a socket mutation (e.g., T366S, L368A, and Y407V as numbered according to the EU numbering scheme), mutations that modulate effector function (e.g., L234A, L235A, and / or P329G (e.g., L234A and L235A) as numbered according to the EU numbering scheme), and / or mutations that increase serum stability or serum half-life (e.g., (i) M252Y, S254T, and T256E as numbered with reference to the EU numbering, or (ii) N434S as numbered with reference to the EU numbering, with or without M428L).

[0447] In some embodiments, the Fc polypeptide can have a knuckle mutation (e.g., T366W as numbered according to the EU numbering scheme) and have at least 85% identity, at least 90% identity, or at least 95% identity with the sequence of any one of SEQ ID NOs: 1 and 4 - 90. In some embodiments, an Fc polypeptide having the sequence of any one of SEQ ID NOs: 1 and 4 - 90 can be modified to have a knuckle mutation.

[0448] In some embodiments, the Fc polypeptide can have a knuckle mutation (e.g., T366W as numbered according to the EU numbering scheme), mutations that modulate effector function (e.g., L234A, L235A, and / or P329G (e.g., L234A and L235A) as numbered according to the EU numbering scheme), and have at least 85% identity, at least 90% identity, or at least 95% identity with the sequence of any one of SEQ ID NOs: 1 and 4 - 90. In some embodiments, an Fc polypeptide having the sequence of any one of SEQ ID NOs: 1 and 4 - 90 can be modified to have a knuckle mutation and mutations that modulate effector function.

[0449] In some embodiments, the Fc polypeptide can have a knuckle mutation (e.g., T366W as numbered according to the EU numbering scheme), mutations that increase serum stability or serum half-life (e.g., (i) M252Y, S254T, and T256E as numbered with reference to the EU numbering, or (ii) N434S as numbered with reference to the EU numbering, with or without M428L), and have at least 85% identity, at least 90% identity, or at least 95% identity with the sequence of any one of SEQ ID NOs: 1 and 4 - 90. In some embodiments, an Fc polypeptide having the sequence of any one of SEQ ID NOs: 1 and 4 - 90 can be modified to have a knuckle mutation and mutations that increase serum stability or serum half-life.

[0450] In some embodiments, the Fc polypeptide can have a staphylococcal nuclease domain mutation (e.g., T366W numbered according to the EU numbering scheme), a mutation that modulates effector function (e.g., L234A, L235A, and / or P329G (e.g., L234A and L235A) numbered according to the EU numbering scheme), a mutation that increases serum stability or serum half-life (e.g., (i) M252Y, S254T, and T256E numbered with reference to the EU numbering, or (ii) N434S numbered with reference to the EU numbering, with or without M428L), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 1 and 4-90. In some embodiments, an Fc polypeptide having the sequence of any one of SEQ ID NOs: 1 and 4-90 can be modified to have a staphylococcal nuclease domain mutation, a mutation that modulates effector function, and a mutation that increases serum stability or serum half-life.

[0451] In some embodiments, the Fc polypeptide can have a papain domain mutation (e.g., T366S, L368A, and Y407V numbered according to the EU numbering scheme) and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 1 and 4-90. In some embodiments, an Fc polypeptide having the sequence of any one of SEQ ID NOs: 1 and 4-90 can be modified to have a papain domain mutation.

[0452] In some embodiments, the Fc polypeptide can have a papain domain mutation (e.g., T366S, L368A, and Y407V numbered according to the EU numbering scheme), a mutation that modulates effector function (e.g., L234A, L235A, and / or P329G (e.g., L234A and L235A) numbered according to the EU numbering scheme), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 1 and 4-90. In some embodiments, an Fc polypeptide having the sequence of any one of SEQ ID NOs: 1 and 4-90 can be modified to have a papain domain mutation and a mutation that modulates effector function.

[0453] In some embodiments, the Fc polypeptide can have CH mutations (e.g., T366S, L368A, and Y407V numbered according to the EU numbering scheme), mutations that increase serum stability or serum half-life (e.g., (i) M252Y, S254T, and T256E numbered with reference to the EU numbering scheme, or (ii) N434S numbered with reference to the EU numbering scheme, with or without M428L), and have at least 85%, at least 90%, or at least 95% identity to the sequence of any one of SEQ ID NOs: 1 and 4 - 90. In some embodiments, an Fc polypeptide having the sequence of any one of SEQ ID NOs: 1 and 4 - 90 can be modified to have CH mutations and mutations that increase serum stability or serum half-life.

[0454] In some embodiments, the Fc polypeptide can have CH mutations (e.g., T366S, L368A, and Y407V numbered according to the EU numbering scheme), mutations that modulate effector function (e.g., L234A, L235A, and / or P329G (e.g., L234A and L235A) numbered according to the EU numbering scheme), mutations that increase serum stability or serum half-life (e.g., (i) M252Y, S254T, and T256E numbered with reference to the EU numbering, or (ii) N434S numbered with reference to the EU numbering, with or without M428L), and have at least 85%, at least 90%, or at least 95% identity to the sequence of any one of SEQ ID NOs: 1 and 4 - 90. In some embodiments, an Fc polypeptide having the sequence of any one of SEQ ID NOs: 1 and 4 - 90 can be modified to have CH mutations, mutations that modulate effector function, and mutations that increase serum stability or serum half-life.

[0455] IV. Exemplary fusion proteins comprising ERT enzyme

[0456] In some aspects, the fusion proteins described herein comprise a first Fc polypeptide that is linked to an enzyme replacement therapy (ERT) enzyme, an ERT enzyme variant, or a catalytically active fragment thereof; and a second Fc polypeptide that forms an Fc dimer with the first Fc polypeptide. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide do not include immunoglobulin heavy and / or light chain variable region sequences or antigen-binding portions thereof. In some embodiments, the ERT enzyme is IDS. In some embodiments, the first Fc polypeptide is a modified Fc polypeptide and / or the second Fc polypeptide is a modified Fc polypeptide. In some embodiments, the second Fc polypeptide is a modified Fc polypeptide. In some embodiments, the modified Fc polypeptide contains one or more modifications that promote its heterodimerization with another Fc polypeptide. In some embodiments, the modified Fc polypeptide contains one or more modifications that reduce effector functions. In some embodiments, the modified Fc polypeptide contains one or more modifications that prolong serum half-life. In some embodiments, the modified Fc polypeptide contains one or more modifications that confer binding to a blood-brain barrier (BBB) receptor, such as the transferrin receptor (TfR).

[0457] In other aspects, the fusion proteins described herein comprise a first polypeptide chain that comprises a modified Fc polypeptide that specifically binds to a BBB receptor such as TfR; and a second polypeptide chain that comprises an Fc polypeptide that dimerizes with the modified Fc polypeptide to form an Fc dimer. The ERT enzyme can be linked to the first or second polypeptide chain. In some embodiments, the ERT enzyme is IDS. In some embodiments, the ERT enzyme is linked to the second polypeptide chain. In some embodiments, the protein comprises two ERT enzymes, each linked to one of the polypeptide chains. In some embodiments, the Fc polypeptide can be a BBB receptor-binding polypeptide that specifically binds to the same BBB receptor as the modified Fc polypeptide in the first polypeptide chain. In some embodiments, the Fc polypeptide does not specifically bind to a BBB receptor.

[0458] In some embodiments, the fusion proteins described herein comprise a first polypeptide chain that comprises a modified Fc polypeptide that specifically binds to TfR; and a second polypeptide chain that comprises an Fc polypeptide, wherein the modified Fc polypeptide dimerizes with the Fc polypeptide to form an Fc dimer. In some embodiments, the ERT enzyme is IDS. In some embodiments, the ERT enzyme is linked to the first polypeptide chain. In some embodiments, the ERT enzyme is linked to the second polypeptide chain. In some embodiments, the Fc polypeptide does not specifically bind to a BBB receptor, such as TfR.

[0459] In some embodiments, the fusion proteins described herein comprise a first polypeptide chain comprising a modified Fc polypeptide that binds to TfR and comprises a T366W (pestle) substitution; and a second polypeptide chain comprising an Fc polypeptide comprising T366S, L368A, and Y407V (mortar) substitutions. In some embodiments, the modified Fc polypeptide and / or the Fc polypeptide further comprise L234A and L235A (LALA) substitutions. In some embodiments, the modified Fc polypeptide and / or the Fc polypeptide further comprise M252Y, S254T, and T256E (YTE) substitutions. In some embodiments, the modified Fc polypeptide and / or the Fc polypeptide further comprise L234A and L235A (LALA) substitutions and M252Y, S254T, and T256E (YTE) substitutions. In some embodiments, the modified Fc polypeptide and / or the Fc polypeptide comprise human IgG1 wild-type residues at positions 234, 235, 252, 254, 256, and 366.

[0460] In some embodiments, the modified Fc polypeptide comprises the pestle, LALA, and YTE mutations as illustrated for any one of SEQ ID NOs: 95-98, 117, 118-123, 130-135, 142-147, 154-159, 166-171, and 178-183 and has at least 85%, at least 90%, or at least 95% identity to the corresponding sequence; or comprises the sequence of any one of SEQ ID NOs: 95-98, 117, 118-123, 130-135, 142-147, 154-159, 166-171, and 178-183. In some embodiments, the Fc polypeptide comprises the mortar, LALA, and YTE mutations as illustrated for any one of SEQ ID NOs: 99-102 and has at least 85%, at least 90%, or at least 95% identity to the corresponding sequence; or comprises the sequence of any one of SEQ ID NOs: 99-102. In some embodiments, the modified Fc polypeptide comprises any one of SEQ ID NOs: 95-98, 117, 118-123, 130-135, 142-147, 154-159, 166-171, and 178-183, and the Fc polypeptide comprises any one of SEQ ID NOs: 99-102. In some embodiments, the N-terminus of the modified Fc polypeptide and / or the Fc polypeptide comprises a portion of the IgG1 hinge region (e.g., DKTHTCPPCP; SEQ ID NO: 111). In some embodiments, the modified Fc polypeptide has at least 85%, at least 90%, or at least 95% identity to any one of SEQ ID NOs: 114, 190, and 191, or comprises the sequence of any one of SEQ ID NOs: 114, 190, and 191.

[0461] In some embodiments, the fusion protein described herein comprises a first polypeptide chain comprising a modified Fc polypeptide that binds to TfR and comprises the T366S, L368A, and Y407V (socket) substitutions; and a second polypeptide chain comprising an Fc polypeptide comprising the T366W (pestle) substitution. In some embodiments, the modified Fc polypeptide and / or the Fc polypeptide further comprise the L234A and L235A (LALA) substitutions. In some embodiments, the modified Fc polypeptide and / or the Fc polypeptide further comprise the M252Y, S254T, and T256E (YTE) substitutions. In some embodiments, the modified Fc polypeptide and / or the Fc polypeptide further comprise the L234A and L235A (LALA) substitutions and the M252Y, S254T, and T256E (YTE) substitutions. In some embodiments, the modified Fc polypeptide and / or the Fc polypeptide comprise human IgG1 wild-type residues at positions 234, 235, 252, 254, 256, and 366.

[0462] In some embodiments, the modified Fc polypeptide comprises the socket, LALA, and YTE mutations as illustrated for any one of SEQ ID NOs: 103-106, 124-129, 136-141, 148-153, 160-165, 172-177, and 184-189 and has at least 85% identity, at least 90% identity, or at least 95% identity to the corresponding sequence; or comprises a sequence of any one of SEQ ID NOs: 103-106, 124-129, 136-141, 148-153, 160-165, 172-177, and 184-189. In some embodiments, the Fc polypeptide comprises the pestle, LALA, and YTE mutations as illustrated for any one of SEQ ID NOs: 107-110 and has at least 85% identity, at least 90% identity, or at least 95% identity to the corresponding sequence; or comprises a sequence of any one of SEQ ID NOs: 107-110. In some embodiments, the modified Fc polypeptide comprises any one of SEQ ID NOs: 103-106, 124-129, 136-141, 148-153, 160-165, 172-177, and 184-189, and the Fc polypeptide comprises any one of SEQ ID NOs: 107-110. In some embodiments, the N-terminus of the modified Fc polypeptide and / or the Fc polypeptide comprises a portion of the IgG1 hinge region (e.g., DKTHTCPPCP; SEQ ID NO: 111).

[0463] In some embodiments, the ERT enzyme such as IDS present in the fusion proteins described herein is linked to a polypeptide chain comprising an Fc polypeptide having at least 85%, at least 90% or at least 95% identity to any one of SEQ ID NOs: 99 - 102, or comprising the sequence of any one of SEQ ID NOs: 99 - 102 (e.g., in a fusion polypeptide). In some embodiments, the ERT enzyme such as IDS is linked to the Fc polypeptide via a linker (e.g., a flexible linker) and / or a hinge region or a portion thereof (e.g., DKTHTCPPCP; SEQ ID NO: 111). In some embodiments, the ERT enzyme comprises an IDS sequence having at least 85%, at least 90% or at least 95% identity to any one of SEQ ID NOs: 112, 192 and 196, or comprising the sequence of any one of SEQ ID NOs: 112, 192 and 196. In some embodiments, the IDS sequence linked to the Fc polypeptide has at least 85%, at least 90% or at least 95% identity to any one of SEQ ID NOs: 113, 115, 193, 194, 197 and 198, or comprises the sequence of any one of SEQ ID NOs: 113, 115, 193, 194, 197 and 198. In some embodiments, the fusion protein comprises a modified Fc polypeptide having at least 85%, at least 90% or at least 95% identity to any one of SEQ ID NOs: 95 - 98, 117, 118 - 123, 130 - 135, 142 - 147, 154 - 159, 166 - 171 and 178 - 183, or comprising the sequence of any one of SEQ ID NOs: 95 - 98, 117, 118 - 123, 130 - 135, 142 - 147, 154 - 159, 166 - 171 and 178 - 183. In some embodiments, the N - terminus of the Fc polypeptide and / or the modified Fc polypeptide comprises a portion of the IgG1 hinge region (e.g., DKTHTCPPCP; SEQ ID NO: 111). In some embodiments, the modified Fc polypeptide has at least 85%, at least 90% or at least 95% identity to any one of SEQ ID NOs: 114, 190 and 191, or comprises the sequence of any one of SEQ ID NOs: 114, 190 and 191.

[0464] In some embodiments, the fusion protein comprises an IDS-Fc fusion polypeptide comprising the sequence of SEQ ID NO:113, and a modified Fc polypeptide comprising the sequence of any one of SEQ ID NO:167 and 190 (e.g., SEQ ID NO:190). In other embodiments, the fusion protein comprises an IDS-Fc fusion polypeptide comprising the sequence of SEQ ID NO:113, and a modified Fc polypeptide comprising the sequence of any one of SEQ ID NO:131 and 191 (e.g., SEQ ID NO:191).

[0465] In some embodiments, the fusion protein comprises an IDS-Fc fusion polypeptide comprising the sequence of SEQ ID NO:193, and a modified Fc polypeptide comprising the sequence of any one of SEQ ID NO:167 and 190 (e.g., SEQ ID NO:190). In other embodiments, the fusion protein comprises an IDS-Fc fusion polypeptide comprising the sequence of SEQ ID NO:193, and a modified Fc polypeptide comprising the sequence of any one of SEQ ID NO:131 and 191 (e.g., SEQ ID NO:191).

[0466] In some embodiments, the fusion protein comprises an IDS-Fc fusion polypeptide comprising the sequence of SEQ ID NO:197, and a modified Fc polypeptide comprising the sequence of any one of SEQ ID NO:167 and 190 (e.g., SEQ ID NO:190). In other embodiments, the fusion protein comprises an IDS-Fc fusion polypeptide comprising the sequence of SEQ ID NO:197, and a modified Fc polypeptide comprising the sequence of any one of SEQ ID NO:131 and 191 (e.g., SEQ ID NO:191).

[0467] In some embodiments, an ERT enzyme such as IDS present in the fusion proteins described herein is linked to a polypeptide chain that comprises an Fc polypeptide having at least 85%, at least 90% or at least 95% identity to any one of SEQ ID NOs: 107 - 110, or comprises the sequence of any one of SEQ ID NOs: 107 - 110 (e.g., in a fusion polypeptide). In some embodiments, an ERT enzyme such as IDS is linked to the Fc polypeptide via a linker (e.g., a flexible linker) and / or a hinge region or a portion thereof (e.g., DKTHTCPPCP; SEQ ID NO: 111). In some embodiments, the ERT enzyme comprises an IDS sequence having at least 85%, at least 90% or at least 95% identity to any one of SEQ ID NOs: 112, 192 and 196, or comprises the sequence of any one of SEQ ID NOs: 112, 192 and 196. In some embodiments, the IDS sequence linked to the Fc polypeptide has at least 85%, at least 90% or at least 95% identity to any one of SEQ ID NOs: 116, 195 and 199, or comprises the sequence of any one of SEQ ID NOs: 116, 195 and 199. In some embodiments, the fusion protein comprises a modified Fc polypeptide having at least 85%, at least 90% or at least 95% identity to any one of SEQ ID NOs: 103 - 106, 124 - 129, 136 - 141, 148 - 153, 160 - 165, 172 - 177 and 184 - 189, or comprises the sequence of any one of SEQ ID NOs: 103 - 106, 124 - 129, 136 - 141, 148 - 153, 160 - 165, 172 - 177 and 184 - 189. In some embodiments, the N-terminus of the Fc polypeptide and / or the modified Fc polypeptide comprises a portion of the IgG1 hinge region (e.g., DKTHTCPPCP; SEQ ID NO: 111).

[0468] In some embodiments, the ERT enzyme, such as IDS, present in the fusion proteins described herein is linked to a polypeptide chain that comprises a modified Fc polypeptide having at least 85%, at least 90%, or at least 95% identity to any one of SEQ ID NOs: 95-98, 117, 118-123, 130-135, 142-147, 154-159, 166-171, and 178-183, or comprises a sequence of any one of SEQ ID NOs: 95-98, 117, 118-123, 130-135, 142-147, 154-159, 166-171, and 178-183 (e.g., in a fusion polypeptide). In some embodiments, the ERT enzyme, such as IDS, is linked to the modified Fc polypeptide via a linker (e.g., a flexible linker) and / or a hinge region or a portion thereof (e.g., DKTHTCPPCP; SEQ ID NO: 111). In some embodiments, the ERT enzyme comprises an IDS sequence having at least 85%, at least 90%, or at least 95% identity to any one of SEQ ID NOs: 112, 192, and 196, or comprises a sequence of any one of SEQ ID NOs: 112, 192, and 196. In some embodiments, the fusion protein comprises an Fc polypeptide having at least 85%, at least 90%, or at least 95% identity to any one of SEQ ID NOs: 99-102, or comprises a sequence of any one of SEQ ID NOs: 99-102. In some embodiments, the N-terminus of the modified Fc polypeptide and / or the Fc polypeptide comprises a portion of the IgG1 hinge region (e.g., DKTHTCPPCP; SEQ ID NO: 111).

[0469] In some embodiments, the ERT enzyme such as IDS present in the fusion proteins described herein is linked to a polypeptide chain that comprises a modified Fc polypeptide having at least 85%, at least 90%, or at least 95% identity to any one of SEQ ID NOs: 103 - 106, 124 - 129, 136 - 141, 148 - 153, 160 - 165, 172 - 177, and 184 - 189, or comprises a sequence of any one of SEQ ID NOs: 103 - 106, 124 - 129, 136 - 141, 148 - 153, 160 - 165, 172 - 177, and 184 - 189 (such as in a fusion polypeptide). In some embodiments, the ERT enzyme such as IDS is linked to the modified Fc polypeptide via a linker (such as a flexible linker) and / or a hinge region or a portion thereof (such as DKTHTCPPCP; SEQ ID NO: 111). In some embodiments, the ERT enzyme comprises an IDS sequence having at least 85%, at least 90%, or at least 95% identity to any one of SEQ ID NOs: 112, 192, and 196, or comprises a sequence of any one of SEQ ID NOs: 112, 192, and 196. In some embodiments, the fusion protein comprises an Fc polypeptide having at least 85%, at least 90%, or at least 95% identity to any one of SEQ ID NOs: 107 - 110, or comprises a sequence of any one of SEQ ID NOs: 107 - 110. In some embodiments, the N - terminus of the modified Fc polypeptide and / or the Fc polypeptide comprises a portion of the IgG1 hinge region (such as DKTHTCPPCP; SEQ ID NO: 111).

[0470] V. ERT Enzymes Linked to Fc Polypeptides

[0471] In some embodiments, the fusion proteins described herein comprise two Fc polypeptides as described herein and one or both of the Fc polypeptides may also comprise a partial or complete hinge region. The hinge region can be from any immunoglobulin subclass or isotype. One exemplary immunoglobulin hinge is the IgG hinge region, such as the IgG1 hinge region, such as the human IgG1 hinge amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO: 93) or a portion thereof (such as DKTHTCPPCP; SEQ ID NO: 111). In some embodiments, the hinge region is in the N - terminal region of the Fc polypeptide.

[0472] In some embodiments, the Fc polypeptide is conjugated to the ERT enzyme via a linker, such as a peptide linker. In some embodiments, the Fc polypeptide is conjugated to the ERT enzyme via a peptide bond or via a peptide linker, such as a fusion polypeptide. The peptide linker can be configured to allow the ERT enzyme to rotate relative to the Fc polypeptide to which it is conjugated; and / or resist protease digestion. The peptide linker can contain natural amino acids, unnatural amino acids, or combinations thereof. In some embodiments, the peptide linker can be a flexible linker, such as containing amino acids, such as Gly, Asn, Ser, Thr, Ala, etc. Such linkers are designed using known parameters and can be of any length and contain any number of repeating units of any length (e.g., repeating units of Gly and Ser residues). For example, the linker can have repeating units, such as two, three, four, five, or more Gly 4 -Ser (SEQ ID NO:201) repeating units or a single Gly 4 -Ser (SEQ ID NO:201). In some embodiments, the peptide linker can include a protease cleavage site, such as can be cleaved by an enzyme present in the central nervous system.

[0473] In some embodiments, the ERT enzyme is conjugated to the N-terminus of the Fc polypeptide, for example, via a Gly 4 -Ser linker (SEQ ID NO:201) or a (Gly 4 -Ser) 2 linker (SEQ ID NO:202). In some embodiments, the Fc polypeptide can contain a hinge sequence or a partial hinge sequence conjugated to the linker or directly conjugated to the ERT enzyme at the N-terminus.

[0474] In some embodiments, the ERT enzyme is conjugated to the C-terminus of the Fc polypeptide, for example, via a Gly 4 -Ser linker (SEQ ID NO:201) or a (Gly 4 -Ser) 2 linker (SEQ ID NO:202). In some embodiments, the C-terminus of the Fc polypeptide is directly conjugated to the ERT enzyme.

[0475] In some embodiments, the ERT enzyme is conjugated to an Fc polypeptide via a chemical crosslinker. Such conjugates can be produced using well-known chemical crosslinking reagents and protocols. For example, there are a large number of chemical crosslinking agents known to those skilled in the art and available for crosslinking polypeptides to agents of interest. For example, the crosslinker is a heterobifunctional crosslinker, which can be used to link molecules in a stepwise manner. Heterobifunctional crosslinkers enable more specific coupling methods to be designed for conjugated proteins, thereby reducing the occurrence of unwanted side reactions such as homoprotein polymers. A variety of heterobifunctional crosslinkers are known in the art, including N-hydroxysuccinimide (NHS) or its water-soluble analogue N-hydroxysulfosuccinimide (sulfo-NHS), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), succinimidyl 4-(p-maleimidophenyl) butyrate (SMPB), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC); 4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)-toluene (SMPT), N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), and succinimidyl 6-[3-(2-pyridyldithio) propionate] hexanoate (LC-SPDP). Those crosslinkers having an N-hydroxysuccinimide moiety are available as N-hydroxysulfosuccinimide analogues, which generally have greater water solubility. Additionally, those crosslinkers having a disulfide bond in the linking chain can alternatively be synthesized as alkyl derivatives to reduce the amount of linker cleavage in vivo. In addition to heterobifunctional crosslinkers, there are many other crosslinkers, including homobifunctional crosslinkers and photoreactive crosslinkers. Disuccinimidyl suberate (DSS), bismaleimidohexane (BMH), and dimethyl pimelimidate.2HCl (DMP) are examples of useful homobifunctional crosslinkers, and bis-[B-(4-azidosalicylamido) ethyl] disulfide (BASED) and N-succinimidyl-6(4'-azido-2'-nitrophenylamino) hexanoate (SANPAH) are examples of useful photoreactive crosslinkers.

[0476] Screening method

[0477] Certain embodiments described herein also provide a method for use as an active screening test agent for LSD treatment, the method comprising:

[0478] 1) contacting a cell with the test agent, wherein the lysosomal storage of the cell is impaired; and

[0479] 2) measuring the concentration of:

[0480] a) A combination of two or more lipids in the cell, wherein the lipid combination is selected from the group consisting of:

[0481] i) BMP;

[0482] ii) GM2 ganglioside and / or GM3 ganglioside;

[0483] iii) GD3;

[0484] iv) GD1a / b; and

[0485] v) GlcCer;

[0486] b) GlcCer in the cell, provided that the test agent is screened for activity as a treatment for MPS; and / or

[0487] c) sTREM2 in the cell,

[0488] wherein a decrease in the concentration of the selected lipid / protein in the cell compared to the concentration of the corresponding lipid / protein in control cells (e.g., healthy cells, e.g., cells without LSD mutations) indicates that the test agent has activity as a treatment for LSD.

[0489] Certain embodiments described herein provide a method for screening a test agent for activity as a treatment for LSD, the method comprising:

[0490] 1) Contacting a cell with the test agent, wherein the lysosomal storage of the cell is impaired; and

[0491] 2) Measuring the concentration of:

[0492] a) A combination of two or more lipids in the cell, wherein the lipid combination is selected from the group consisting of:

[0493] i) BMP;

[0494] ii) GM2 ganglioside and / or GM3 ganglioside;

[0495] iii) GD3;

[0496] iv) GD1a / b; and

[0497] v) GlcCer;

[0498] b) GlcCer in the cell, provided that the test agent is screened for activity as a treatment for MPS;

[0499] c) Nf-L in the cell; and / or

[0500] d) sTREM2 in said cells,

[0501] wherein a decrease in the concentration of a selected lipid / protein in said cells as compared to the concentration of the corresponding lipid / protein in control cells (e.g., healthy cells, e.g., cells without LSD mutations) indicates that the test agent has activity as a treatment for LSD.

[0502] In certain embodiments, the method includes measuring the concentration of sTREM2.

[0503] In certain embodiments, the method includes measuring the concentration of Nf-L.

[0504] In certain embodiments, the method includes measuring the concentration of GlcCer, wherein the test agent is screened for activity as a treatment for MPS.

[0505] In certain embodiments, the method includes measuring the concentration of a combination of two or more lipids, such as the combinations described herein.

[0506] In certain embodiments, the method includes measuring the concentration of one or more lipids and the concentration of sTREM2.

[0507] In certain embodiments, the method includes measuring the concentration of one or more lipids and the concentration of Nf-L.

[0508] In certain embodiments, the method includes measuring the concentration of sTREM2 and the concentration of Nf-L.

[0509] In certain embodiments, the method includes measuring the concentration of one or more lipids, the concentration of sTREM2, and the concentration of Nf-L.

[0510] In certain embodiments, the cells are from tissue. For example, in certain embodiments, the cells are cells from the brain, liver, kidney, lung, or spleen. In certain embodiments, the cells are brain cells or cells derived from brain cells. In an embodiment, the cells are cells obtained from CSF. In an embodiment, the cells are cells obtained from serum.

[0511] In certain embodiments, the concentration of the lipid / protein is measured using an assay described herein (e.g., mass spectrometry).

[0512] Method for isolating an enriched population of CNS cells

[0513] Provided herein are methods for isolating a population of enriched CNS cell types (e.g., a population of enriched neurons, astrocytes, or microglia) from brain tissue.

[0514] Accordingly, certain embodiments provide a method of sorting CNS cell populations from a tissue sample, the method comprising:

[0515] (a) contacting the tissue sample with a primary antibody to a neuronal marker, a primary antibody to an astrocyte marker, a primary antibody to a microglia marker, a primary antibody to an endothelial marker, and a primary antibody to an oligodendrocyte marker, wherein each primary antibody is uniquely labeled to provide a labeled tissue sample; and

[0516] (b) sorting the cells in the labeled tissue sample by flow cytometry,

[0517] wherein the method provides distinct cell populations of neurons, astrocytes, and microglia.

[0518] As used herein, the term "distinct cell population" refers to a physically separated cell population that is enriched in a particular CNS cell type (e.g., neurons, astrocytes, microglia).

[0519] As used herein, the term "neuronal marker" refers to a protein or peptide that is preferentially expressed by neurons in the CNS. In certain embodiments, at least about 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 99.5% of the neuronal cells present in the CNS express the neuronal marker. In certain embodiments, less than about 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% of the non-neuronal cells present in the CNS express the neuronal marker. In certain embodiments, non-neuronal cell types present in the CNS do not express the neuronal marker. In certain embodiments, the neuronal marker is Thy1 (see, e.g., UniProtKB P01831 (mouse)).

[0520] As used herein, the term "primary antibody to a neuronal marker" or "anti-neuronal marker antibody" refers to an antibody that can bind to a neuronal marker with sufficient affinity such that the antibody can be used to sort neurons from a mixed cell population using flow cytometry. In one embodiment, the anti-neuronal marker antibody binds to an irrelevant protein to an extent that is less than about 10% of the binding of the antibody to the neuronal marker, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the dissociation constant (Kd) of the antibody that binds to the neuronal marker is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9M to 10 -13 M). In certain embodiments, the neuronal marker primary antibody is an anti-Thy1 antibody (such as the anti-Thy1 antibody used herein).

[0521] As used herein, the term "astrocyte marker" refers to a protein or peptide preferentially expressed by astrocytes in the CNS. In certain embodiments, at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or 99.5% of the astrocytes present in the CNS express the astrocyte marker. In certain embodiments, less than about 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.5% of the non-astrocyte cells present in the CNS express the astrocyte marker. In certain embodiments, non-astrocyte cell types present in the CNS do not express the astrocyte marker. In certain embodiments, the astrocyte marker is excitatory amino acid transporter 2 (EAAT2) (see, e.g., UniProtKB P43006 (mouse)). In certain embodiments, the astrocyte marker is a glycosylated surface molecule recognized by an anti-astrocyte surface antigen 2 (ACSA-2) antibody (see, e.g., Kantzer et al., 2017, Glia, 65:990-1004).

[0522] As used herein, the term "astrocyte marker primary antibody" or "anti-astrocyte marker antibody" refers to an antibody capable of binding to an astrocyte marker with sufficient affinity such that the antibody can be used to sort astrocytes from a mixed cell population using flow cytometry. In one embodiment, the degree of binding of the anti-astrocyte marker antibody to an irrelevant protein is less than about 10% of the binding of the antibody to the astrocyte marker, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the dissociation constant (Kd) of the binding of the antibody to the astrocyte marker is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM or ≤0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M). In certain embodiments, the astrocyte marker primary antibody is an anti-EAAT2 antibody (such as the anti-EAAT2 antibody used herein). In certain embodiments, the astrocyte marker primary antibody is an anti-ACSA-2 antibody (such as ACSA-2-PE (Miltenyi Biotec 130-102-365)).

[0523] As used herein, the term "microglial marker" refers to a protein or peptide that is preferentially expressed by microglial cells within the CNS. In certain embodiments, at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or 99.5% of the microglial cells present in the CNS express the microglial marker. In certain embodiments, less than about 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.5% of the non-microglial cells present in the CNS express the microglial marker. In certain embodiments, non-microglial cell types present in the CNS do not express the microglial marker. In certain embodiments, the microglial marker is CD11b (see, e.g., UniProtKB P05555 (mouse)).

[0524] As used herein, the term "microglial marker primary antibody" or "anti-microglial marker antibody" refers to an antibody that can bind to the microglial marker with sufficient affinity such that the antibody can be used to sort microglial cells from a mixed cell population using flow cytometry. In one embodiment, the degree of binding of the anti-microglial marker antibody to an irrelevant protein is less than about 10% of the binding of the antibody to the microglial marker, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the dissociation constant (Kd) of the binding of the antibody to the microglial marker is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM or ≤0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M). In certain embodiments, the microglial marker primary antibody is an anti-CD11b antibody (e.g., the anti-CD11b antibody used herein).

[0525] As used herein, the term "endothelial marker" refers to a protein or peptide that is preferentially expressed by endothelial cells within the CNS. In certain embodiments, at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or 99.5% of the endothelial cells present in the CNS express the endothelial cell marker. In certain embodiments, less than about 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.5% of the non-endothelial cells present in the CNS express the endothelial cell marker. In certain embodiments, non-endothelial cell types present in the CNS do not express the endothelial cell marker. In certain embodiments, the endothelial cell marker is CD31 (see, e.g., UniProtKB Q08481 (mouse)).

[0526] As used herein, the term "endothelial marker primary antibody" or "anti-endothelial cell marker antibody" refers to an antibody that can bind to an endothelial cell marker with sufficient affinity such that the antibody can be used to sort endothelial cells from a mixed cell population using flow cytometry. In one embodiment, the degree of binding of the anti-endothelial cell marker antibody to an irrelevant protein is less than about 10% of the binding of the antibody to the endothelial cell marker, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the dissociation constant (Kd) of the binding of the antibody to the endothelial cell marker is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M). In certain embodiments, the endothelial marker primary antibody is an anti-CD31 antibody (e.g., the anti-CD-31 antibody as used herein).

[0527] As used herein, the term "oligodendrocyte marker" refers to a protein or peptide that is preferentially expressed by oligodendrocytes within the CNS. In certain embodiments, at least about 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 99.5% of the oligodendrocytes present in the CNS express the oligodendrocyte marker. In certain embodiments, less than about 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% of the non-oligodendrocytes present in the CNS express the oligodendrocyte marker. In certain embodiments, non-oligodendrocyte types present in the CNS do not express the oligodendrocyte marker. In certain embodiments, the oligodendrocyte marker is a membrane lipid marker. In certain embodiments, the oligodendrocyte marker is a lipid that is enriched on mature oligodendrocytes, such as galactocerebroside (GalCer).

[0528] As used herein, the term "oligodendrocyte marker primary antibody" or "anti-oligodendrocyte marker antibody" refers to an antibody that can bind to an oligodendrocyte marker with sufficient affinity such that the antibody can be used to sort oligodendrocytes from a mixed cell population using flow cytometry. In one embodiment, the degree of binding of the anti-oligodendrocyte marker antibody to an irrelevant protein is less than about 10% of the binding of the antibody to the oligodendrocyte marker, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the dissociation constant (Kd) of the binding of the antibody to the oligodendrocyte marker is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM or ≤0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M). In certain embodiments, the oligodendrocyte marker primary antibody is an anti-O1 antibody that reacts with a membrane lipid marker (e.g., GalCer) (e.g., the anti-O1 antibody used herein).

[0529] In certain embodiments, a tissue sample is contacted with an anti-Thy1 antibody, an anti-EAAT2 antibody, an anti-CD11b antibody, an anti-CD31 antibody, and an anti-O1 antibody. In certain embodiments, a tissue sample is contacted with an anti-Thy1 antibody, an anti-ACSA-2 antibody, an anti-CD11b antibody, an anti-CD31 antibody, and an anti-O1 antibody. In certain embodiments, the primary antibodies are included in a composition and the tissue sample is contacted with the composition. In certain embodiments, each primary antibody is uniquely labeled with a label (e.g., a fluorescent label) suitable for sorting by flow cytometry (i.e., each antibody contains a different label). In certain embodiments, the tissue sample is further contacted with a viability dye that can be used to distinguish live cells from non-live cells by flow cytometry (e.g., the fixable viability dye BV510). In certain embodiments, the tissue sample is contacted with the viability dye simultaneously or sequentially with the primary antibodies. In certain other embodiments, the viability dye is included in the composition containing the primary antibodies and the tissue sample is contacted with the composition containing the primary antibodies and the viability dye.

[0530] In certain embodiments, the cells present in the tissue sample are dissociated prior to contact with the viability dye, the primary antibodies, and / or the composition containing the primary antibodies with or without the viability dye.

[0531] In certain embodiments, the tissue sample is contacted with the primary antibodies under conditions suitable for the antibodies to bind to their corresponding markers and label the cells. In certain embodiments, the labeled tissue sample prior to sorting by flow cytometry contains labeled Thy1+ Cells, labeled EAAT2 + Cells, labeled CD11b + Cells, labeled CD31 + Cells and labeled O1 + Cells. In certain embodiments, the tissue sample labeled prior to sorting by flow cytometry comprises labeled Thy1 + Cells, labeled ACSA-2 + Cells, labeled CD11b + Cells, labeled CD31 + Cells and labeled O1 + Cells. In certain embodiments, the cells are further labeled with a viability dye.

[0532] In certain embodiments, the cells present in the tissue sample are sorted by flow cytometry into a non-viable cell population and a viable cell population (e.g., with a viability dye).

[0533] In certain embodiments, the cells present in the tissue sample are sorted by flow cytometry into an O1 + cell population and an O1 - cell population.

[0534] In certain embodiments, the cells present in the tissue sample are sorted by flow cytometry into a CD31 + cell population and a CD31 - cell population.

[0535] In certain embodiments, the cells present in the tissue sample are sorted by flow cytometry into a Thy + cell population and a Thy - cell population.

[0536] In certain embodiments, the cells present in the tissue sample are sorted by flow cytometry into an EAAT2 + cell population and an EAAT2 - cell population.

[0537] In certain embodiments, the cells present in the tissue sample are sorted by flow cytometry into an ACSA-2 + cell population and an ACSA-2 - cell population.

[0538] In certain embodiments, the cells present in the tissue sample are sorted by flow cytometry into a CD11b + cell population and a CD11b - cell population.

[0539] In certain embodiments, the cells present in the tissue sample are sorted by flow cytometry into viable, O1 - , CD31 - , Thy1 + , EAAT2 - cell populations (i.e., neuronal cells).

[0540] In certain embodiments, the cells present in the tissue sample are sorted by flow cytometry into viable, O1 - , CD31 - , Thy1 - , EAAT2 + cell populations (i.e., astrocytes).

[0541] In certain embodiments, the cells present in the tissue sample are sorted by flow cytometry into viable, O1 - , CD31 - , CD11b + cell populations (i.e., microglia).

[0542] In certain embodiments, the cells present in the tissue sample are sorted by flow cytometry into viable, O1 - , CD31 - , Thy1 + , ACSA-2 - cell populations (i.e., neuronal cells).

[0543] In certain embodiments, the cells present in the tissue sample are sorted by flow cytometry into viable, O1 - , CD31 - , Thy1 - , ACSA-2 + cell populations (i.e., astrocytes).

[0544] In certain embodiments, the cells present in the tissue sample are sorted by flow cytometry into non-viable cell populations and viable cell populations (e.g., with viability dyes). In certain embodiments, the viable cell populations are further sorted into O1 - , CD31 - cell populations (i.e., oligodendrocytes and endothelial cells) and O1 + cell populations. In certain embodiments, O1

[0545] In certain embodiments, the cells present in the tissue sample are sorted by flow cytometry into non-viable cell populations and viable cell populations (e.g., with viability dyes). In certain embodiments, the viable cell populations are further sorted into O1 + , CD31 + cell populations (i.e., oligodendrocytes and endothelial cells) and O1 - , CD31 - cell populations. In certain embodiments, O1- , CD31 - The cell population was further sorted into viable, O1 - , CD31 - , Thy1 + , EAAT2 - cell (i.e., neuron cell) population; viable, O1 - , CD31 - , Thy1 - , EAAT2 + cell (i.e., astrocyte) population; and viable, O1 - , CD31 - , CD11b + cell (i.e., microglia) population.

[0546] In certain embodiments, the cells present in the tissue sample are sorted by flow cytometry into a non-viable cell population and a viable cell population (e.g., with a viability dye). In certain embodiments, the viable cell population is further sorted into an O1 + , CD31 + cell (i.e., oligodendrocyte and endothelial cell) population and an O1 - , CD31 - cell population. In certain embodiments, the O1 - , CD31 - cell population is further sorted into viable, O1 - , CD31 - , Thy1 + , ACSA-2 - cell (i.e., neuron cell) population; viable, O1 - , CD31 - , Thy1 - , ACSA-2 + cell (i.e., astrocyte) population; and viable, O1 - , CD31 - , CD11b + cell (i.e., microglia) population.

[0547] In certain embodiments, the cells present in the tissue sample are sorted by flow cytometry into a non-viable cell population and a viable cell population (e.g., with a viability dye). In certain embodiments, the viable cell population is further sorted into a CD31 + cell (i.e., endothelial cell) population and a CD31 - cell population. In certain embodiments, the CD31 - cell population is further sorted into CD31 - , Thy1 +, EAAT2 - Cell population; CD31 - , Thy1 - , EAAT2 + Cell population; and CD31 - , CD11b + Cell population. In certain embodiments, CD31 - , Thy1 + , EAAT2 - Cells; CD31 - , Thy1 - , EAAT2 + Cells; and CD31 - , CD11b + Cell populations are further sorted to remove O1 + Cells (i.e., oligodendrocytes), thereby providing viable, O1 - , CD31 - , Thy1 + , EAAT2 - Cell population (i.e., neuronal cells); viable, O1 - , CD31 - , Thy1 - , EAAT2 + Cell population (i.e., astrocyte cells); and viable, O1 - , CD31 - , CD11b + Cell population (i.e., microglial cells).

[0548] In certain embodiments, the cells present in a tissue sample are sorted by flow cytometry into a non-viable cell population and a viable cell population (e.g., with a viability dye). In certain embodiments, the viable cell population is further sorted into a CD31 + Cell population (i.e., endothelial cells) and a CD31 - Cell population. In certain embodiments, the CD31 - Cell population is further sorted into a CD31 - , Thy1 + , ACSA-2 - Cell population; CD31 - , Thy1 - , ACSA-2 + Cell population; and CD31 - , CD11b + Cell population. In certain embodiments, CD31 - , Thy1 + , ACSA-2 - Cell population; CD31- 、Thy1 - 、ACSA-2 + cell populations; and CD31 - 、CD11b + The cell populations were further sorted to remove O1 + cells (i.e., oligodendrocytes), thereby providing live, O1 - 、CD31 - 、Thy1 + 、ACSA-2 - cell populations (i.e., neuronal cells); live, O1 - 、CD31 - 、Thy1 - 、ACSA-2 + cell populations (i.e., astrocytes); and live, O1 - 、CD31 - 、CD11b + cell populations (i.e., microglial cells).

[0549] In certain embodiments, the microglial cell population is sorted based on the marker profile O1 - / CD31 - / CD11b + sorting.

[0550] In certain embodiments, the astrocyte cell population is sorted based on the marker profile O1 - / CD31 - / Thy1 - / EAAT2 + sorting. In certain embodiments, the astrocyte cell population is sorted based on the marker profile O1 - / CD31 - / Thy1 - / ACSA-2 + sorting.

[0551] In certain embodiments, the neuronal population is sorted based on the marker profile O1 - / CD31 - / Thy1 + / EAAT2 - sorting. In certain embodiments, the neuronal population is sorted based on the marker profile O1 - / CD31 - / Thy1 + / ACSA-2 - sorting.

[0552] In certain embodiments, the sorted and enriched neuronal cells (e.g., live, O1 - 、CD31- 、Thy1 + 、EAAT2 - cells or live, O1 - 、CD31 - 、Thy1 + 、ACSA-2 - The population of cells) contains less than about 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less non-neuronal cells. In certain embodiments, the sorted and enriched neuronal cells (e.g., live, O1 - 、CD31 - 、Thy1 + 、EAAT2 - cells or live, O1 - 、CD31 - 、Thy1 + 、ACSA-2 - cells) population contains no non-neuronal cells.

[0553] In certain embodiments, the sorted and enriched astrocytes (e.g., live, O1 - 、CD31 - 、Thy1 - 、EAAT2 + cells or live, O1 - 、CD31 - 、Thy1 - 、ACSA-2 + cells) population contains less than about 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less non-astrocyte cells. In certain embodiments, the sorted and enriched astrocytes (e.g., live, O1 - 、CD31 - 、Thy1 - 、EAAT2 + cells or live, O1 - 、CD31 - 、Thy1 - 、ACSA-2 + cells) population contains no non-astrocyte cells.

[0554] In certain embodiments, the sorted and enriched microglia (e.g., live, O1 - 、CD31 - 、CD11b+ The (cell) population contains less than about 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less non - microglial cells. In certain embodiments, the sorted and enriched microglial cells (e.g., live, O1 - , CD31 - , CD11b + cell) population contains no non - microglial cells.

[0555] In certain embodiments, one or more (e.g., 1, 2, or 3) of the enriched cell populations are analyzed to quantify metabolites or nucleic acids. In certain embodiments, the enriched neuronal cell population is analyzed to quantify metabolites or nucleic acids. In certain embodiments, the enriched astrocyte population is analyzed to quantify metabolites or nucleic acids. In certain embodiments, the enriched microglial cell population is analyzed to quantify metabolites or nucleic acids. In certain embodiments, the enriched neuronal, astrocyte, and microglial cell populations are analyzed to quantify metabolites or nucleic acids. In certain embodiments, one or more of the enriched cell populations are analyzed to quantify metabolites. In certain embodiments, one or more of the enriched cell populations are analyzed to quantify more than one metabolite (e.g., 2, 3, 4, 5, 10, 25, 50 or more). In certain embodiments, one or more of the enriched cell populations are analyzed to quantify nucleic acids. In certain embodiments, one or more of the enriched cell populations are analyzed to quantify more than one nucleic acid (e.g., 2, 3, 4, 5, 10, 25, 50 or more). In certain embodiments, one or more of the enriched cell populations are analyzed to quantify metabolites and nucleic acids. In certain embodiments, one or more of the enriched cell populations are analyzed to quantify more than one metabolite and more than one nucleic acid.

[0556] As used herein, the term metabolite includes macromolecules that are typically broken down by lysosomes. For example, in certain embodiments, the metabolite is a glycosaminoglycan (GAG) species, such as the GAG species described herein (e.g., D0S0, D0A0, or D0a4). In certain embodiments, the metabolite is a lipid species, such as gangliosides, glycosphingolipids (e.g., glucosylceramide), galactosylceramide, and bis(monoacylglycerol) phosphate (BMP) species (e.g., the species described herein). In certain embodiments, the metabolite is a BMP, GlcCer, GD3, GD1a / b, GM2, and / or GM3 species (e.g., as described herein). In certain embodiments, a combination of metabolites is quantified, such as a combination of lipids described herein. Metabolites can be quantified using methods known in the art. For example, metabolites can be quantified using liquid chromatography mass spectrometry (LCMS) assays (see, e.g., the Examples).

[0557] The nucleic acid can be, for example, RNA or DNA, such as genomic DNA, RNA transcribed from genomic DNA, or cDNA generated from RNA. In certain embodiments, the nucleic acid species is RNA. In certain embodiments, the nucleic acid species is DNA. In certain embodiments, the nucleic acid species is genomic DNA. Methods for quantifying nucleic acid species are known in the art. For example, such methods include, but are not limited to, polymerase chain reaction (PCR), including quantitative PCR (qPCR) and real-time quantitative reverse transcription PCR (qRT-PCR); RNAseq; RNA blot analysis, expression microarray analysis; next-generation sequencing (NGS); and fluorescence in situ hybridization (FISH). In certain embodiments, the nucleic acid species is quantified using the assays described herein.

[0558] In certain embodiments, one or more enriched cell populations are analyzed to quantify sTREM2. In certain embodiments, an enriched microglial cell population is analyzed to quantify sTREM2. sTREM2 can be quantified using methods known in the art. For example, sTREM2 can be quantified using the assays described in the Examples.

[0559] In certain embodiments, one or more enriched cell populations are analyzed to quantify Nf-L. In certain embodiments, an enriched neuronal cell population is analyzed to quantify Nf-L. Nf-L can be quantified using methods known in the art. For example, Nf-L can be quantified using the assays described in the Examples.

[0560] In certain embodiments, one or more enriched cell populations are analyzed to quantify a therapeutically administered agent. In certain embodiments, an enriched neuronal cell population is analyzed to quantify a therapeutically administered agent. In certain embodiments, an enriched astrocyte population is analyzed to quantify a therapeutically administered agent. In certain embodiments, an enriched microglial cell population is analyzed to quantify a therapeutically administered agent. In certain embodiments, enriched neuronal, astrocyte, and microglial cell populations are analyzed to quantify a therapeutically administered agent. In certain embodiments, the therapeutically administered agent is an agent capable of reducing one or more symptoms associated with LSD. In certain embodiments, the therapeutically administered agent is ETV:IDS. Methods for quantifying the therapeutically administered agent are known in the art and are described herein. By way of example, the therapeutically administered agent can be quantified by the assays described in the Examples.

[0561] Certain embodiments provide a plurality of CNS cells comprising three physically separated cell populations, wherein:

[0562] 1) The first cell population comprises an enriched O1 - / CD31 - / CD11b + cell population;

[0563] 2) The second cell population comprises an enriched O1 - / CD31 - / Thy1 - / EAAT2 + cell population or an enriched O1 - / CD31 - / Thy1 - / ACSA-2 + cell population; and

[0564] 3) The third cell population comprises an enriched O1 - / CD31 - / Thy1 + / EAAT2 - cell population or an enriched O1 - / CD31 - / Thy1 + / ACSA-2 - cell population.

[0565] Corrected cells and related methods

[0566] As described herein, LSD is caused by a deficiency of certain lysosomal enzymes, resulting in the accumulation of metabolites within certain cell types (e.g., certain CNS cells). This accumulation can be corrected by contacting the affected cells with certain therapeutic agents (e.g., the therapeutic agents described herein). For example, the accumulation of metabolites within cells having a lysosomal enzyme deficiency (e.g., CNS cells) can be reduced by contacting the cells with a fusion protein comprising an enzyme replacement therapy (ERT) enzyme linked to an Fc polypeptide.

[0567] Accordingly, certain embodiments provide a corrected CNS cell comprising a lysosomal enzyme deficiency that causes the accumulation of metabolites within the cell, wherein the correction is effected by contacting the cell with a protein comprising: (i) a first Fc polypeptide linked to the lysosomal enzyme, and (ii) a second Fc polypeptide that forms an Fc dimer with the first Fc polypeptide, wherein the protein is capable of binding to the transferrin receptor (TfR), and wherein the correction is a reduction in the accumulation of the metabolites. In certain embodiments, the corrected CNS cell is a human cell. In certain embodiments, the corrected CNS cell is a non-human cell. In certain embodiments, the cell is an isolated or purified corrected CNS cell.

[0568] Certain embodiments also provide a corrected CNS cell comprising a reduced accumulation of metabolites, wherein a CNS cell comprising a lysosomal enzyme deficiency that causes the accumulation of the metabolites within the cell is contacted with a protein comprising:

[0569] (i) a first Fc polypeptide linked to the lysosomal enzyme; and

[0570] (ii) a second Fc polypeptide that forms an Fc dimer with the first Fc polypeptide, wherein the protein is capable of binding to the transferrin receptor (TfR),

[0571] to provide the corrected CNS cell comprising a reduced accumulation of the metabolites.

[0572] Certain embodiments also provide a corrected CNS cell produced by the methods described herein, such as the methods described below.

[0573] Certain embodiments also provide a method of correcting a CNS cell having a lysosomal enzyme deficiency that causes an accumulation of a metabolite in the cell, the method comprising contacting the cell with a protein comprising: (i) a first Fc polypeptide linked to the lysosomal enzyme; and (ii) a second Fc polypeptide that forms an Fc dimer with the first Fc polypeptide, wherein the protein is capable of binding to the transferrin receptor (TfR) to provide a corrected CNS cell having a reduced accumulation of the metabolite.

[0574] Certain embodiments also provide a method of reducing the accumulation of a metabolite in a CNS cell having a lysosomal enzyme deficiency, the method comprising contacting the cell with a protein comprising: (i) a first Fc polypeptide linked to the lysosomal enzyme; and (ii) a second Fc polypeptide that forms an Fc dimer with the first Fc polypeptide, wherein the protein is capable of binding to the transferrin receptor (TfR).

[0575] In certain embodiments, the cell is contacted with the protein in vitro, ex vivo, or in vivo. In certain embodiments, the cell is contacted with the protein in vitro. In certain embodiments, the cell is contacted with the protein ex vivo. In certain embodiments, the cell is contacted with the protein in vivo (i.e., via administration of the protein). In certain embodiments, the cell is from a tissue sample and has been sorted by the methods described herein. In certain embodiments, the cell is contacted with the protein in vivo and before sorting. In certain other embodiments, the cell is contacted with the protein in vitro before or after sorting.

[0576] In certain embodiments, the first Fc polypeptide linked to the lysosomal enzyme is the Fc polypeptide described herein. In certain embodiments, the lysosomal enzyme is iduronate-2-sulfatase (IDS), or a catalytically active variant or fragment of wild-type IDS, such as wild-type human IDS. In certain embodiments, the second Fc polypeptide is the polypeptide described herein. In certain embodiments, the protein is ETV:IDS.

[0577] In certain embodiments, when assayed under the same affinity assay conditions, the protein has an affinity for TfR that is at least 5-fold, 10-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold or greater compared to an irrelevant target. In certain embodiments, the protein binds to TfR with an affinity of from about 50 nM to about 350 nM. In certain embodiments, the protein binds to TfR with an affinity of about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 275 nM, about 300 nM, about 325 nM or about 350 nM.

[0578] In certain embodiments, the metabolite is a GAG substance, such as the GAG substances described herein (e.g., D0S0, D0A0 or D0a4). In certain embodiments, the metabolite is a lipid substance, such as gangliosides, glycosphingolipids (e.g., glucosylceramide), galactosylceramide and bis(monoacylglycerol) phosphate (BMP) substances (e.g., the substances described herein). In certain embodiments, the metabolite is a BMP, GlcCer, GD3, GD1a / b, GM2 and / or GM3 substance (e.g., as described herein). In certain embodiments, the accumulation of a combination of metabolites is reduced, such as a combination of lipids described herein. The amount of metabolite accumulation in cells can be quantified using methods known in the art. For example, the metabolite can be quantified using a liquid chromatography mass spectrometry (LCMS) assay (see, e.g., the Examples).

[0579] In certain embodiments, the accumulation of at least one metabolite in the cell is reduced by at least about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In certain embodiments, the accumulation of at least one metabolite in the cell is reduced to the level in control cells (e.g., corresponding cells that do not contain a lysosomal enzyme deficiency). In certain embodiments, the accumulation of multiple metabolites is reduced (e.g., 2 or more metabolites, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50 or more).

[0580] In certain embodiments, the CNS cells are selected from the group consisting of neurons, astrocytes, and microglia. In certain embodiments, the CNS cells are neurons. In certain embodiments, the CNS cells are astrocytes. In certain embodiments, the CNS cells are microglia.

[0581] Certain embodiments

[0582] Embodiment 1. A method for detecting one or more biomarkers in a subject having a lysosomal storage disorder (LSD), the method comprising:

[0583] 1) Measuring the concentration of a combination of two or more lipids in a sample from the subject, wherein the combination of lipids is selected from the group consisting of:

[0584] a) Bis(monoacylglycerol) phosphate (BMP);

[0585] b) GM2 ganglioside and / or GM3 ganglioside;

[0586] c) GD3 ganglioside;

[0587] d) GD1a / b ganglioside; and

[0588] e) Glucosylceramide (GlcCer);

[0589] 2) Measuring the concentration of GlcCer in a sample from the subject, provided that the LSD is a mucopolysaccharidosis (MPS) disorder;

[0590] 3) Measuring the concentration of neurofilament light chain (Nf-L) in a sample from the subject; and / or

[0591] 4) Measuring the concentration of soluble triggering receptor expressed on myeloid cells 2 (sTREM2) in a sample from the subject.

[0592] Embodiment 2. A method for evaluating the therapeutic efficacy in a subject having an LSD, the method comprising:

[0593] 1) Measuring the concentration of a combination of two or more lipids in a sample obtained from the subject after administration of the treatment, wherein the combination of lipids is selected from the group consisting of:

[0594] a) BMP;

[0595] b) GM2 ganglioside and / or GM3 ganglioside;

[0596] c) GD3;

[0597] d) GD1a / b; and

[0598] e) GlcCer;

[0599] 2) Measuring the concentration of GlcCer in a sample obtained from the subject after administration of the treatment, provided that the LSD is an MPS disorder;

[0600] 3) Measuring the concentration of Nf-L in a sample obtained from the subject after administration of the treatment; and / or

[0601] 4) Measuring the concentration of sTREM2 in a sample obtained from the subject after administration of the treatment;

[0602] wherein a decrease in the concentration of the lipid / protein in the sample obtained from the subject after administration of the treatment, compared to the concentration of the selected lipid / protein in a sample obtained from the subject before administration of the treatment, is correlated with treatment efficacy.

[0603] Embodiment 3. A method of identifying a subject with an LSD as a treatment candidate, the method comprising:

[0604] 1) Measuring the concentration of a combination of two or more lipids in a sample from the subject, wherein the combination of lipids is selected from the group consisting of:

[0605] a) BMP;

[0606] b) GM2 ganglioside and / or GM3 ganglioside;

[0607] c) GD3;

[0608] d) GD1a / b; and

[0609] e) GlcCer;

[0610] 2) Measuring the concentration of GlcCer in a sample from the subject, provided that the LSD is an MPS disorder;

[0611] 3) Measuring the concentration of Nf-L in a sample from the subject; and / or

[0612] 4) Measuring the concentration of sTREM2 in a sample from the subject;

[0613] wherein the subject can be identified as a treatment candidate only if the concentration of the selected lipid / protein in the sample from the subject is at least as high as a control value.

[0614] Embodiment 4. The method of any one of Embodiments 1-3, the method further comprising administering an LSD treatment to the subject.

[0615] Embodiment 5. The method of any one of Embodiments 1-4, the method further comprising adjusting the treatment regimen of the subject.

[0616] Embodiment 6. A method of treating LSD in a subject, the method comprising:

[0617] 1) Administering an LSD treatment to the subject;

[0618] 2) Measuring the concentration of:

[0619] a) A combination of two or more lipids in a sample from the subject, wherein the lipid combination is selected from the group consisting of:

[0620] i) BMP;

[0621] ii) GM2 ganglioside and / or GM3 ganglioside;

[0622] iii) GD3;

[0623] iv) GD1a / b; and

[0624] v) GlcCer;

[0625] b) GlcCer in a sample from the subject, provided that the LSD is an MPS disorder;

[0626] c) Nf-L in a sample from the subject; and / or

[0627] d) sTREM2 in a sample from the subject; and

[0628] 3) Adjusting the dose of the LSD treatment based on the concentration of the selected lipid / protein in the sample from the subject as compared to a control value.

[0629] Embodiment 7. The method of any one of Embodiments 1-6, the method comprising measuring the concentration of sTREM2.

[0630] Embodiment 8. The method of any one of Embodiments 1-6, the method comprising measuring the concentration of Nf-L.

[0631] Embodiment 9. The method of any one of Embodiments 1-6, the method comprising measuring the concentration of GlcCer, wherein the LSD is an MPS disorder.

[0632] Embodiment 10. The method of any one of Embodiments 1-6, the method comprising measuring the concentration of a combination of two or more lipids.

[0633] Embodiment 11. The method according to any one of Embodiments 1-6, the method comprising measuring the concentration of one or more lipids and the concentration of sTREM2.

[0634] Embodiment 12. The method according to any one of Embodiments 1-6, the method comprising measuring the concentration of one or more lipids and the concentration of Nf-L.

[0635] Embodiment 13. The method according to any one of Embodiments 1-12, wherein the sample is a tissue sample, a serum sample or a cerebrospinal fluid sample.

[0636] Embodiment 14. The method according to Embodiment 13, wherein the sample is a tissue sample.

[0637] Embodiment 15. The method according to Embodiment 14, wherein the tissue is the brain, liver, kidney, lung or spleen.

[0638] Embodiment 16. The method according to Embodiment 13, wherein the sample is a serum sample.

[0639] Embodiment 17. The method according to Embodiment 13, wherein the sample is a cerebrospinal fluid sample.

[0640] Embodiment 18. A method for treating LSD in a subject, the method comprising administering an LSD treatment to the subject, wherein the subject has or is determined to have:

[0641] 1) A combination of two or more lipids with increased concentration compared to a control, wherein the combination of lipids is selected from the group consisting of:

[0642] a) BMP;

[0643] b) GM2 ganglioside and / or GM3 ganglioside;

[0644] c) GD3;

[0645] d) GD1a / b; and

[0646] e) GlcCer;

[0647] 2) Increased concentration of GlcCer, provided that the LSD is an MPS disorder;

[0648] 3) Increased concentration of Nf-L; and / or

[0649] 4) Increased concentration of sTREM2.

[0650] Embodiment 19. The method according to Embodiment 18, wherein the subject has or is determined to have an increased concentration of sTREM2.

[0651] Embodiment 20. The method of Embodiment 18, wherein the subject has or is determined to have an increased concentration of Nf-L.

[0652] Embodiment 21. The method of Embodiment 18, wherein the subject has or is determined to have an increased concentration of GlcCer, and wherein the LSD is an MPS disorder.

[0653] Embodiment 22. The method of Embodiment 18, wherein the subject has or is determined to have a combination of two or more lipids at increased concentrations.

[0654] Embodiment 23. The method of Embodiment 18, wherein the subject has or is determined to have one or more lipids at increased concentrations and sTREM2 at increased concentration.

[0655] Embodiment 24. The method of Embodiment 18, wherein the subject has or is determined to have one or more lipids at increased concentrations and Nf-L at increased concentration.

[0656] Embodiment 25. The method of any one of Embodiments 1-24, wherein the combination comprises BMP.

[0657] Embodiment 26. The method of any one of Embodiments 1-25, wherein the combination comprises GlcCer.

[0658] Embodiment 27. The method of any one of Embodiments 1-26, wherein the combination comprises GD3.

[0659] Embodiment 28. The method of any one of Embodiments 1-27, wherein the combination comprises GD1a / b.

[0660] Embodiment 29. The method of any one of Embodiments 1-28, wherein the combination comprises GM2.

[0661] Embodiment 30. The method of any one of Embodiments 1-29, wherein the combination comprises GM3.

[0662] Embodiment 31. The method of any one of Embodiments 1-24, wherein the combination comprises: BMP and GlcCer; BMP and GD3; BMP and GD1a / b; BMP and GM2; BMP and GM3; GlcCer and GD3; GlcCer and GD1a / b; GlcCer and GM2; GlcCer and GM3; GD3 and GD1a / b; GD3 and GM2; GD3 and GM3; GD1a / b and GM2; GD1a / b and GM3; BMP, GlcCer and GD3; BMP, GlcCer and GD1a / b; BMP, GlcCer and GM2; BMP, GlcCer and GM3; BMP, GD3 and GD1a / b; BMP, GD3 and GM2; BMP, GD3 and GM3; BMP, GD1a / b and GM2; BMP, GD1a / b and GM3; BMP, GM2 and GM3; GlcCer, GD3 and GD1a / b; GlcCer, GD3 and GM2; GlcCer, GD3 and GM3; GlcCer, GD1a / b and GM2; GlcCer, GD1a / b and GM3; GlcCer, GM2 and GM3; GD3, GD1a / b and GM2; GD3, GD1a / b and GM3; GD3, GM2 and GM3; GD1a / b, GM2 and GM3; BMP, GlcCer, GD3 and GD1a / b; BMP, GlcCer, GD3 and GM2; BMP, GlcCer, GD3 and GM3; BMP, GlcCer, GD1a / b and GM2; BMP, GlcCer, GD1a / b and GM3; BMP, GlcCer, GM2 and GM3; BMP, GD3, GD1a / b and GM2; BMP, GD3, GD1a / b and GM3; BMP, GD3, GM2 and GM3; BMP, GD1a / b, GM2 and GM3; GlcCer, GD3, GD1a / b and GM2; GlcCer, GD3, GD1a / b and GM3; GlcCer, GD3, GM2 and GM3; GlcCer, GD1a / b, GM2 and GM3; GD3, GD1a / b, GM2 and GM3; BMP, GlcCer, GD3, GD1a / b and GM2; BMP, GlcCer, GD3, GD1a / b and GM3; BMP, GD3, GD1a / b, GM2 and GM3; BMP, GlcCer, GD3, GM2 and GM3; BMP, GlcCer, GD1a / b, GM2 and GM3; GlcCer, GD3, GD1a / b, GM2 and GM3; or BMP, GlcCer, GD3, GD1 / b, GM2 and GM3.

[0663] Embodiment 32. The method of any one of Embodiments 1-24, wherein the combination comprises: BMP and GlcCer; BMP and GD3; BMP and GD1a / b; BMP and GM2; BMP and GM3; GlcCer and GD3; GlcCer and GD1a / b; GlcCer and GM2; GlcCer and GM3; GD3 and GD1a / b; GD3 and GM2; GD3 and GM3; GD1a / b and GM2; or GD1a / b and GM3.

[0664] Embodiment 33. The method of any one of Embodiments 1-24, wherein the combination comprises: BMP, GlcCer and GD3; BMP, GlcCer and GD1a / b; BMP, GlcCer and GM2; BMP, GlcCer and GM3; BMP, GD3 and GD1a / b; BMP, GD3 and GM2; BMP, GD3 and GM3; BMP, GD1a / b and GM2; BMP, GD1a / b and GM3; BMP, GM2 and GM3; GlcCer, GD3 and GD1a / b; GlcCer, GD3 and GM2; GlcCer, GD3 and GM3; GlcCer, GD1a / b and GM2; GlcCer, GD1a / b and GM3; GlcCer, GM2 and GM3; GD3, GD1a / b and GM2; GD3, GD1a / b and GM3; GD3, GM2 and GM3; or GD1a / b, GM2 and GM3.

[0665] Embodiment 34. The method of any one of Embodiments 1-24, wherein the combination comprises: BMP, GlcCer, GD3 and GD1a / b; BMP, GlcCer, GD3 and GM2; BMP, GlcCer, GD3 and GM3; BMP, GlcCer, GD1a / b and GM2; BMP, GlcCer, GD1a / b and GM3; BMP, GlcCer, GM2 and GM3; BMP, GD3, GD1a / b and GM2; BMP, GD3, GD1a / b and GM3; BMP, GD3, GM2 and GM3; BMP, GD1a / b, GM2 and GM3; GlcCer, GD3, GD1a / b and GM2; GlcCer, GD3, GD1a / b and GM3; GlcCer, GD3, GM2 and GM3; GlcCer, GD1a / b, GM2 and GM3; or GD3, GD1a / b, GM2 and GM3.

[0666] Embodiment 35. The method of any one of embodiments 1-24, wherein the combination comprises: BMP, GlcCer, GD3, GD1a / b, and GM2; BMP, GlcCer, GD3, GD1a / b, and GM3; BMP, GD3, GD1a / b, GM2, and GM3; BMP, GlcCer, GD3, GM2, and GM3; BMP, GlcCer, GD1a / b, GM2, and GM3; or GlcCer, GD3, GD1a / b, GM2, and GM3.

[0667] Embodiment 36. The method of any one of embodiments 1-24, wherein the combination comprises: BMP, GlcCer, GD3, GD1 / b, GM2, and GM3.

[0668] Embodiment 37. The method of any one of embodiments 1-36, wherein the LSD is an MPS disorder.

[0669] Embodiment 38. The method of embodiment 37, wherein the MPS disorder is Hunter's syndrome.

[0670] Embodiment 39. The method of any one of embodiments 2-38, wherein the LSD treatment comprises hematopoietic stem cell transplantation (HSCT), enzyme replacement therapy (ERT), substrate reduction therapy, chaperone therapy, and / or gene therapy.

[0671] Embodiment 40. The method of embodiment 39, wherein the LSD treatment comprises ERT.

[0672] Embodiment 41. The method of embodiment 40, wherein the ERT targets the brain.

[0673] Embodiment 42. The method of embodiment 40, wherein the LSD treatment is a protein comprising:

[0674] (a) a first Fc polypeptide linked to an enzyme replacement therapy (ERT) enzyme, an ERT enzyme variant, or a catalytically active fragment thereof; and

[0675] (b) a second Fc polypeptide that forms an Fc dimer with the first Fc polypeptide,

[0676] wherein the first Fc polypeptide and / or the second Fc polypeptide do not include immunoglobulin heavy and / or light chain variable region sequences or antigen-binding portions thereof.

[0677] Embodiment 43. The method of embodiment 42, wherein the ERT enzyme is iduronate-2-sulfatase (IDS), an IDS variant, or a catalytically active fragment thereof.

[0678] Embodiment 44. The method of embodiment 42, wherein the first Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 113, 193, and 197, and the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 114.

[0679] Embodiment 45. The method of embodiment 42, wherein the first Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 113, 193, and 197, and the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 131.

[0680] Embodiment 46. The method of embodiment 42, wherein the first Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 113, 193, and 197, and the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 167.

[0681] Embodiment 47. The method of embodiment 42, wherein the first Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 113, 193, and 197, and the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 190.

[0682] Embodiment 48. The method of embodiment 42, wherein the first Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 113, 193, and 197, and the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 191.

[0683] Embodiment 49. The method of embodiment 42, wherein the first Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 113, 193, and 197, and the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 117.

[0684] Embodiment 50. The method of embodiment 42, wherein the first Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 113, 193, and 197, and the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 130.

[0685] Embodiment 51. The method of embodiment 42, wherein the first Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 113, 193, and 197, and the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 132.

[0686] Embodiment 52. The method of embodiment 42, wherein the first Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 113, 193, and 197, and the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 166.

[0687] Embodiment 53. The method of embodiment 42, wherein the first Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 113, 193, and 197, and the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 168.

[0688] Embodiment 54. A method for active screening of a test agent for LSD treatment, the method comprising:

[0689] 1) contacting a cell with the test agent, wherein the lysosomal storage of the cell is impaired; and

[0690] 2) measuring the concentration of:

[0691] a) a combination of two or more lipids in the cell, wherein the lipid combination is selected from the group consisting of:

[0692] i) BMP;

[0693] ii) GM2 ganglioside and / or GM3 ganglioside;

[0694] iii) GD3;

[0695] iv) GD1a / b; and

[0696] v) GlcCer;

[0697] b) GlcCer in the cell, provided that the test agent is actively screened for MPS treatment;

[0698] c) Nf-L in the cell; and / or

[0699] d) sTREM2 in the cell;

[0700] wherein a decrease in the concentration of the selected lipid / protein in the cell compared to the concentration of the corresponding lipid / protein in control cells indicates that the test agent has activity as an LSD treatment.

[0701] Embodiment 55. The method of embodiment 54, the method comprising measuring the concentration of sTREM2.

[0702] Embodiment 56. The method of embodiment 54, the method comprising measuring the concentration of Nf-L.

[0703] Embodiment 57. The method of embodiment 54, the method comprising measuring the concentration of GlcCer.

[0704] Embodiment 58. The method of embodiment 54, the method comprising measuring the concentration of a combination of two or more lipids.

[0705] Embodiment 59. The method of embodiment 54, the method comprising measuring the concentration of one or more lipids and the concentration of sTREM2.

[0706] Embodiment 60. The method of embodiment 54, the method comprising measuring the concentration of one or more lipids and the concentration of Nf-L.

[0707] Embodiment 61. The method of any one of embodiments 54-60, wherein the cell is a brain cell.

[0708] Embodiment 62. A corrected CNS cell, the CNS cell comprising a lysosomal enzyme deficiency that causes the accumulation of intracellular metabolites, wherein the cell is contacted with a protein comprising: (i) a first Fc polypeptide linked to the lysosomal enzyme, and (ii) a second Fc polypeptide that forms an Fc dimer with the first Fc polypeptide, wherein the protein is capable of binding to the transferrin receptor (TfR), and wherein the correction is to reduce the accumulation of the metabolite.

[0709] Embodiment 63. A corrected CNS cell, the CNS cell comprising a reduced accumulation of metabolites, wherein a CNS cell comprising a lysosomal enzyme deficiency that causes the accumulation of the metabolite intracellularly is contacted with a protein comprising:

[0710] (i) a first Fc polypeptide linked to the lysosomal enzyme; and

[0711] (ii) a second Fc polypeptide that forms an Fc dimer with the first Fc polypeptide, wherein the protein is capable of binding to the transferrin receptor (TfR),

[0712] to provide the corrected CNS cell comprising a reduced accumulation of the metabolite.

[0713] Embodiment 64. The CNS cell of embodiment 62 or 63, wherein the protein binds to TfR with an affinity of about 50 nM to about 350 nM.

[0714] Embodiment 65. The CNS cell of any one of embodiments 62-64, wherein the enzyme is iduronate-2-sulfatase (IDS) or a catalytically active variant thereof.

[0715] CNS cells of any one of embodiments 62-65, wherein the metabolite is glycosaminoglycan (GAG) and / or lysosomal lipid.

[0716] Embodiment 67. CNS cells of any one of embodiments 62-65, wherein the metabolite is glycosaminoglycan (GAG).

[0717] Embodiment 68. CNS cells of any one of embodiments 62-65, wherein the metabolite is lysosomal lipid.

[0718] Embodiment 69. The CNS cells of embodiment 68, wherein the lysosomal lipid is selected from the group consisting of ganglioside, glucosylceramide, galactosylceramide, and bis(monoacylglycerol) phosphate (BMP).

[0719] Embodiment 70. CNS cells of any one of embodiments 62-69, wherein the CNS cells are selected from the group consisting of neurons, astrocytes, and microglia.

[0720] Embodiment 71. A method for sorting a population of CNS cells from a tissue sample, the method comprising:

[0721] (a) contacting the tissue sample with a primary antibody against a neuronal marker, a primary antibody against an astrocyte marker, a primary antibody against a microglial marker, a primary antibody against an endothelial marker, and a primary antibody against an oligodendrocyte marker, wherein each primary antibody is uniquely labeled to provide a labeled tissue sample; and

[0722] (b) sorting the cells in the labeled tissue sample by flow cytometry,

[0723] wherein the method provides unique cell populations of neurons, astrocytes, and microglia.

[0724] Embodiment 72. The method of embodiment 71, wherein the primary antibody against the neuronal marker is an anti-Thy1 antibody.

[0725] Embodiment 73. The method of embodiment 71 or 72, wherein the primary antibody against the microglial marker is an anti-CD11b antibody.

[0726] Embodiment 74. The method of any one of embodiments 71-73, wherein the primary antibody against the astrocyte marker is selected from the group consisting of an anti-EAAT2 antibody and an anti-astrocyte surface antigen-2 (ACSA-2) antibody.

[0727] Embodiment 75. The method of any one of embodiments 71-74, wherein the primary antibody against the endothelial marker is an anti-CD31 antibody.

[0728] Embodiment 76. The method of any one of Embodiments 71-75, wherein the oligodendrocyte marker primary antibody is an anti-O1 antibody.

[0729] Embodiment 77. The method of any one of Embodiments 71-76, the method further comprising contacting the tissue sample with a vital dye.

[0730] Embodiment 78. The method of any one of Embodiments 71-77, the method providing a unique population of microglia comprising less than about 20% non-microglial cells, a unique population of astrocytes comprising less than about 20% non-astroglial cells, and / or a unique population of neurons comprising less than about 20% non-neuronal cells.

[0731] Embodiment 79. The method of Embodiment 78, the method providing a unique population of microglia comprising less than about 20% non-microglial cells.

[0732] Embodiment 80. The method of Embodiment 78 or 79, the method providing a unique population of astrocytes comprising less than about 20% non-astroglial cells.

[0733] Embodiment 81. The method of any one of Embodiments 78-80, the method providing a unique population of neurons comprising less than about 20% non-neuronal cells.

[0734] Embodiment 82. The method of any one of Embodiments 71-81, wherein the microglia population is sorted based on the marker profile O1 - / CD31 - / CD11b + ; the astrocyte population is sorted based on the marker profile O1 - / CD31 - / Thy1 - / EAAT2 + or O1 - / CD31 - / Thy1 - / ACSA-2 + ; and / or the neuron population is sorted based on the marker profile O1 - / CD31 - / Thy1 + / EAAT2 - or O1 - / CD31 - / Thy1 + / ACSA-2 - sorting.

[0735] Embodiment 83. The method of Embodiment 82, wherein the microglial cell population is based on the marker profile O1 - / CD31 - / CD11b + Sorted.

[0736] Embodiment 84. The method of Embodiment 82 or 83, wherein the astrocyte cell population is based on the marker profile O1 - / CD31 - / Thy1 - / EAAT2 + or O1 - / CD31 - / Thy1 - / ACSA-2 + Sorted.

[0737] Embodiment 85. The method of any one of Embodiments 82-84, wherein the neuronal cell population is based on the marker profile O1 - / CD31 - / Thy1 + / EAAT2 - or O1 - / CD31 - / Thy1 + / ACSA-2 - Sorted.

[0738] Embodiment 86. The method of any one of Embodiments 71-85, wherein the enriched cell population is analyzed to quantify sTREM2, Nf-L, metabolites, and / or nucleic acid substances.

[0739] Embodiment 87. The method of any one of Embodiments 71-85, wherein the enriched cell population is analyzed to quantify metabolite or nucleic acid substances.

[0740] Embodiment 88. The method of Embodiment 87, wherein the metabolite is a glycosaminoglycan (GAG) substance.

[0741] Embodiment 89. The method of Embodiment 87, wherein the metabolite is a lipid substance.

[0742] Embodiment 90. The method of Embodiment 89, wherein the lipid substance is selected from the group consisting of gangliosides, glucosylceramides, galactosylceramides, and bis(monoacylglycerol) phosphate (BMP).

[0743] Embodiment 91. The method of Embodiment 87, wherein the nucleic acid substance is selected from RNA, DNA, and genomic DNA.

[0744] Embodiment 92. The method of any one of Embodiments 71-91, wherein the enriched cell population is analyzed to quantify sTREM2.

[0745] Embodiment 93. The method of any one of Embodiments 71-92, wherein the enriched cell population is analyzed to quantify Nf-L.

[0746] Embodiment 94. The method of any one of Embodiments 71-93, wherein the enriched cell population is analyzed to quantify the administered therapeutic agent.

[0747] Embodiment 95. The method of Embodiment 94, wherein the administered therapeutic agent is ETV:IDS.

[0748] Certain Definitions

[0749] The term "control" or "control sample" refers to any sample suitable for the detection technique employed. A control sample may contain the product of the detection technique employed or the material to be tested. Additionally, the control may be a positive or negative control.

[0750] The terms "subject", "individual", and "patient", which are used interchangeably herein, refer to a mammal, including but not limited to a human, non-human primate, rodent (e.g., rat, mouse, and guinea pig), rabbit, cow, pig, horse, and other mammalian species. In one embodiment, the subject is a human.

[0751] The term "pharmaceutically acceptable excipient" refers to an inactive pharmaceutical ingredient that is biologically or pharmacologically suitable for use in a human or animal, such as but not limited to a buffer, carrier, or preservative.

[0752] The term "administer" refers to a method of delivering an agent (e.g., an LSD therapeutic agent, such as the ETV therapy described herein), compound, or composition (e.g., a pharmaceutical composition) to a desired biological site of action. These methods include but are not limited to oral, topical, parenteral, intravenous, intradermal, intramuscular, intrathecal, colonic, rectal, or intraperitoneal delivery. In one embodiment, the polypeptide described herein is administered intravenously.

[0753] As used herein, "treatment" (and its grammatical variations such as "treat" or "treating") refers to a clinical intervention that modifies the natural course of an individual being treated and can be performed for prophylaxis or during the course of a clinical pathology. Desirable treatment effects include but are not limited to: preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, improving or alleviating the disease state, and relieving or improving the prognosis.

[0754] The phrase "effective amount" means an amount of a compound described herein that achieves one of the following: (i) treats or prevents a particular disease, disorder, or condition; (ii) alleviates, ameliorates, or eliminates one or more symptoms of a particular disease, disorder, or condition; or (iii) prevents or delays the onset of one or more symptoms of a particular disease, disorder, or condition described herein.

[0755] The "therapeutically effective amount" of a substance / molecule disclosed herein may vary depending on various factors such as the disease state, the age, sex, and weight of the individual, and the ability of the substance / molecule to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount that results in a therapeutic benefit that far outweighs any toxicity or detrimental effects of the substance / molecule. A "prophylactically effective amount" is an amount that is effective in achieving the desired prophylactic result at the required dosage and for the required period of time. Generally but not necessarily, since prophylactic doses are administered to subjects before or at an earlier stage of a disease, a prophylactically effective amount will be less than a therapeutically effective amount.

[0756] The terms "obtaining a sample from a patient", "obtained from a patient", and similar expressions are used to refer to obtaining a sample directly from a patient and obtaining a sample indirectly from a patient through an intermediary (e.g., obtaining a sample from a courier who obtained the sample from a nurse who obtained the sample from the patient).

[0757] "Enzyme replacement therapy enzyme" or "ERT enzyme" refers to an enzyme that is deficient in lysosomal storage diseases. An "ERT enzyme variant" refers to a functional variant of a wild-type ERT enzyme or a fragment thereof, including allelic and splicing variants, wherein, for example, when assayed under the same conditions, the ERT enzyme variant has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of its corresponding wild-type ERT enzyme or fragment thereof. A "catalytically active fragment" of an ERT enzyme refers to a portion of a full-length ERT enzyme or a variant thereof, wherein, for example, when assayed under the same conditions, the catalytically active fragment has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding full-length ERT enzyme or variant thereof.

[0758] As used herein, "iduronate sulfatase", "iduronic acid-2-sulfatase", or "IDS" refers to iduronate 2-sulfatase (EC 3.1.6.13), an enzyme involved in the lysosomal degradation of the glycosaminoglycans heparan sulfate and dermatan sulfate. IDS deficiency is associated with mucopolysaccharidosis II (also known as Hunter syndrome). As used herein, the term "IDS" as a component of a protein comprising an Fc polypeptide has catalytic activity and encompasses functional variants of wild-type IDS or fragments thereof, including allelic and splice variants. The sequence of human IDS isoform I, the human sequence designated as the canonical sequence, is available under UniProt entry P22304 and is encoded by the human IDS gene located at Xq28. The full-length sequence is provided as SEQ ID NO:91. As used herein, a "mature" IDS sequence refers to a polypeptide chain form that lacks the signal and propeptide sequences of the naturally occurring full-length polypeptide chain. The amino acid sequence of mature human IDS polypeptide is provided as SEQ ID NO:92, which corresponds to amino acids 34 - 550 of the full-length human sequence. As used herein, a "truncated" IDS sequence refers to a catalytically active fragment of the naturally occurring full-length polypeptide chain. The amino acid sequence of an exemplary truncated human IDS polypeptide is provided as SEQ ID NO:112, which corresponds to amino acids 26 - 550 of the full-length human sequence. The structure of human IDS has been well characterized. An exemplary structure is available under PDB accession number 5FQL. This structure is also described in Nat.Comm. 8:15786 doi:10.1038 / ncomms15786, 2017. Non-human primate IDS sequences have also been described, including those of chimpanzee (UniProt entry K7BKV4) and rhesus macaque (UniProt entry H9FTX2). The mouse IDS sequence is available under Uniprot entry Q08890. When measured under the same conditions, for example, an IDS variant has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding wild-type IDS or its fragment. When measured under the same conditions, for example, a catalytically active IDS fragment has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding full-length IDS or its variant.

[0759] As used herein, "transferrin receptor" or "TfR" refers to transferrin receptor protein 1. The human transferrin receptor 1 polypeptide sequence is shown in SEQ ID NO:94. Transferrin receptor protein 1 sequences from other species are also known (e.g., chimpanzee, accession number XP_003310238.1; rhesus macaque, NP_001244232.1; dog, NP_001003111.1; bovine, NP_001193506.1; mouse, NP_035768.1; rat, NP_073203.1; and chicken NP_990587.1). The term "transferrin receptor" also encompasses exemplary reference sequences, such as allelic variants of the human sequence, which are encoded by genes at the chromosomal locus of transferrin receptor protein 1. The full-length transferrin receptor protein includes a short N-terminal intracellular region, a transmembrane region, and a large extracellular domain. The extracellular domain is characterized by three domains: a protease-like domain, a helical domain, and an apical domain. The apical domain sequence of human transferrin receptor 1 is shown in SEQ ID NO:200.

[0760] As used herein, "fusion protein" or "[ERT enzyme]-Fc fusion protein" refers to a dimeric protein that comprises a first Fc polypeptide (i.e., "[ERT]-Fc fusion polypeptide") linked (e.g., fused) to an ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof; and a second Fc polypeptide that forms an Fc dimer with the first Fc polypeptide. The second Fc polypeptide may also be linked (e.g., fused) to an ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof. The first Fc polypeptide and / or the second Fc polypeptide may be linked to the ERT enzyme, the ERT enzyme variant, or the catalytically active fragment thereof by a peptide bond or through a polypeptide linker. The first Fc polypeptide and / or the second Fc polypeptide may be a modified Fc polypeptide that contains one or more modifications that promote heterodimerization with another Fc polypeptide. The first Fc polypeptide and / or the second Fc polypeptide may be a modified Fc polypeptide that contains one or more modifications that confer binding to the transferrin receptor. The first Fc polypeptide and / or the second Fc polypeptide may be a modified Fc polypeptide that contains one or more modifications that reduce effector functions. The first Fc polypeptide and / or the second Fc polypeptide may be a modified Fc polypeptide that contains one or more modifications that extend the serum half-life.

[0761] As used herein, "fusion polypeptide" or "[ERT enzyme]-Fc fusion polypeptide" refers to an Fc polypeptide linked (e.g., fused) to an ERT enzyme, an ERT enzyme variant, or a catalytically active fragment thereof. The Fc polypeptide can be linked to the ERT enzyme, ERT enzyme variant, or catalytically active fragment thereof by a peptide bond or through a polypeptide linker. The Fc polypeptide can be a modified Fc polypeptide containing one or more modifications that promote heterodimerization with another Fc polypeptide. The Fc polypeptide can be a modified Fc polypeptide containing one or more modifications that confer binding to the transferrin receptor. The Fc polypeptide can be a modified Fc polypeptide containing one or more modifications that reduce effector functions. The Fc polypeptide can be a modified Fc polypeptide containing one or more modifications that extend the serum half-life.

[0762] As used herein, the term "Fc polypeptide" refers to the C-terminal region of a naturally occurring immunoglobulin heavy chain polypeptide, characterized by Ig folds as domains. The Fc polypeptide contains a constant region sequence comprising at least the CH2 domain and / or CH3 domain, and can contain at least a portion of the hinge region. Generally, the Fc polypeptide does not contain a variable region.

[0763] "Modified Fc polypeptide" refers to an Fc polypeptide having at least one mutation (e.g., substitution, deletion, or insertion) compared to the wild-type immunoglobulin heavy chain Fc polypeptide sequence, but retaining the overall Ig fold or structure of the native Fc polypeptide.

[0764] The term "FcRn" refers to the neonatal Fc receptor. Binding of the Fc polypeptide to FcRn reduces the clearance rate of the Fc polypeptide and increases its serum half-life. The human FcRn protein is a heterodimer composed of a protein similar to the major histocompatibility (MHC) class I protein of approximately 50 kDa and β2-microglobulin of approximately 15 kDa.

[0765] As used herein, "FcRn binding site" refers to the region of the Fc polypeptide that binds to FcRn. In human IgG, using the EU index numbering, the FcRn binding site includes T250, L251, M252, I253, S254, R255, T256, T307, E380, M428, H433, N434, H435, and Y436. These positions correspond to positions 20 to 26, 77, 150, 198, and 203 to 206 of SEQ ID NO:1.

[0766] As used herein, "native FcRn binding site" refers to the region of the Fc polypeptide that binds to FcRn and has the same amino acid sequence as the region of the native Fc polypeptide that binds to FcRn.

[0767] As used herein, the terms "CH3 domain" and "CH2 domain" refer to immunoglobulin constant region domain polypeptides. For the purposes of this application, the CH3 domain polypeptide refers to the amino acid segment from approximately position 341 to approximately position 447 according to the EU numbering, and the CH2 domain polypeptide refers to the amino acid segment from approximately position 231 to approximately position 340 and excluding the hinge region sequence according to the EU numbering scheme. The CH2 and CH3 domain polypeptides can also be numbered according to the IMGT (ImMunoGeneTics) numbering scheme, where according to the IMGT Scientific chart numbering (IMGT website), the CH2 domain is numbered 1-110, and the CH3 domain is numbered 1-107. The CH2 and CH3 domains are part of the immunoglobulin Fc region. The Fc region refers to the amino acid segment from approximately position 231 to approximately position 447 according to the EU numbering scheme, but as used herein, can include at least a portion of the hinge region of the antibody. An exemplary hinge region sequence is the human IgG1 hinge sequence EPKSCDKTHTCPPCP (SEQ ID NO:93).

[0768] "Naturally occurring", "natural" or "wild-type" are used to describe an object that can be found in nature and is different from that which is artificially produced. For example, a nucleotide sequence present in an organism (including a virus) that can be isolated from a natural source and not intentionally modified in the laboratory is naturally occurring. In addition, "wild-type" refers to a normal gene or organism found in nature without any known mutations. For example, the terms "wild-type", "natural" and "naturally occurring" with respect to the CH3 or CH2 domain are used herein to refer to a domain having a naturally occurring sequence.

[0769] As used herein, the terms "mutant" and "variant" with respect to a mutant polypeptide or mutant polynucleotide are used interchangeably. Variants with respect to a given wild-type CH3 or CH2 domain reference sequence can include naturally occurring allelic variants. A CH3 or CH2 domain that is "non-naturally" occurring refers to a variant or mutant domain that does not exist in cells in nature and is produced by genetic modification (e.g., using genetic engineering techniques or mutagenesis techniques) of a native CH3 domain or CH2 domain polynucleotide or polypeptide. A "variant" includes any domain that contains at least one amino acid mutation relative to the wild-type. Mutations can include substitutions, insertions and deletions.

[0770] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that act in a manner similar to naturally occurring amino acids.

[0771] Naturally occurring amino acids are those encoded by the genetic code and those that are post-translationally modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. "Amino acid analogs" are compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. Such analogs have a modified R group (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as the naturally occurring amino acid. "Amino acid mimetics" are compounds that have a structure different from the general chemical structure of amino acids, but act in a manner similar to naturally occurring amino acids.

[0772] Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Stereoisomers of naturally occurring α-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

[0773] Amino acids can be represented herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature.

[0774] The terms "polypeptide" and "peptide" are used interchangeably herein and refer to polymers of amino acid residues in a single chain. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to both naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Amino acid polymers can contain solely L-amino acids, solely D-amino acids, or mixtures of L- and D-amino acids.

[0775] As used herein, the term "protein" refers to a dimer (i.e., two) or multimer (i.e., three or more) of polypeptides or single-chain polypeptides. The single-chain polypeptides of a protein can be joined by covalent bonds, such as disulfide bonds, or non-covalent interactions.

[0776] The terms "conservative substitution", "conservative mutation", or "conservative modified variant" refer to an alteration that results in the replacement of an amino acid with another amino acid that can be classified as having similar characteristics. Examples of classes of conservative amino acid groups defined in this way can include: "charged / polar groups", including Glu (glutamic acid or E), Asp (aspartic acid or D), Asn (asparagine or N), Gln (glutamine or Q), Lys (lysine or K), Arg (arginine or R), and His (histidine or H); "aromatic groups", including Phe (phenylalanine or F), Tyr (tyrosine or Y), Trp (tryptophan or W), and (histidine or H); and "aliphatic groups", including Gly (glycine or G), Ala (alanine or A), Val (valine or V), Leu (leucine or L), Ile (isoleucine or I), Met (methionine or M), Ser (serine or S), Thr (threonine or T), and Cys (cysteine or C). Subgroups can also be identified within each group. For example, the group of charged or polar amino acids can be subdivided into subgroups, including: a "positively charged subgroup" containing Lys, Arg, and His; a "negatively charged subgroup" containing Glu and Asp; and a "polar subgroup" containing Asn and Gln. In another example, the aromatic or cyclic group can be subdivided into subgroups, including: a "nitrogenous ring subgroup" containing Pro, His, and Trp; and a "phenyl subgroup" containing Phe and Tyr. In another example, the aliphatic group can be subdivided into subgroups, such as an "aliphatic non-polar subgroup" containing Val, Leu, Gly, and Ala; and an "aliphatic weakly polar subgroup" containing Met, Ser, Thr, and Cys. Examples of classes of conservative mutations include amino acid substitutions within the above-mentioned subgroups, such as but not limited to: Lys substituting for Arg and vice versa, thus maintaining a positive charge; Glu substituting for Asp and vice versa, thus maintaining a negative charge; Ser substituting for Thr and vice versa, thus maintaining a free -OH; and Gln substituting for Asn and vice versa, thus maintaining a free -NH 2 . In some embodiments, hydrophobic amino acid substitutions are made, for example, for naturally occurring hydrophobic amino acids in the active site, to maintain hydrophobicity.

[0777] In the context of two or more polypeptide sequences, the term "identity" or "percent identity" refers to the extent to which two or more sequences or subsequences are identical or have a specified percentage of identical amino acid residues (e.g., at least 60% identity, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or greater) in a particular region when compared and aligned for maximum correspondence over a comparison window or specified region, as measured using a sequence comparison algorithm or by manual alignment and visual inspection.

[0778] For polypeptide sequence comparison, usually an amino acid sequence is used as a reference sequence to which a candidate sequence is compared. Alignment can be performed using various methods available to those skilled in the art, such as visual alignment or using known algorithms with publicly available software to achieve maximum alignment. Such programs include the BLAST program, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.), or Megalign (DNASTAR). The parameters for alignment to achieve maximum alignment can be determined by those skilled in the art. For the purposes of this application, for polypeptide sequence comparison, the two protein sequences are aligned using the BLASTP algorithm standard protein BLAST with default parameters.

[0779] When used in the context of identifying a given amino acid residue in a polypeptide sequence, the terms "corresponding to", "determined with reference to...", or "numbered with reference to..." refer to the position of the residue in the designated reference sequence when the given amino acid sequence is maximally aligned and compared with the reference sequence. Thus, for example, when an amino acid residue in a modified Fc polypeptide is aligned with the amino acids in SEQ ID NO:1 when optimally aligned with SEQ ID NO:1, that residue "corresponds to" the amino acid in SEQ ID NO:1. The polypeptide aligned with the reference sequence need not be the same length as the reference sequence.

[0780] The terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to nucleotide chains of any length and include DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or any substrate that can be incorporated into the chain by DNA or RNA polymerase. Polynucleotides can contain modified nucleotides such as methylated nucleotides and their analogs. Examples of polynucleotides contemplated herein include single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, and hybrid molecules having mixtures of single-stranded and double-stranded DNA and RNA.

[0781] As used herein, "binding affinity" refers to the strength of the non-covalent interaction between two molecules, e.g., a single binding site on a polypeptide and its bound target, such as the transferrin receptor. Thus, for example, unless otherwise indicated or clear from the context, the term can refer to a 1:1 interaction between a polypeptide and its target. Binding affinity can be quantified by measuring the equilibrium dissociation constant (K D ), where K D refers to the dissociation rate constant (k d , time -1 ) divided by the association rate constant (k a , time-1 M -1 )。K D can be determined, for example, by using the following methods to measure complex formation and dissociation kinetics: surface plasmon resonance (SPR) methods, such as the Biacore TM system; kinetic exclusion assays, such as and biolayer interferometry (e.g., using the platform). As used herein, "binding affinity" includes not only formal binding affinity, such as the binding affinity reflecting a 1:1 interaction between a polypeptide and its target, but also the apparent affinity that can reflect avid binding for calculating K D .

[0782] As used herein, the terms "specifically bind" or "selectively bind" to a target such as TfR when referring to an engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody as described herein refer to a binding reaction in which the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody binds to the target with greater affinity, greater avidity, and / or for a longer duration than it binds to a structurally different target. In a typical embodiment, when measured under the same affinity assay conditions, the affinity of an engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody for a specific target (such as TfR) is at least 5-fold, 10-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or greater than that for an unrelated target. As used herein, the terms "specifically bind", "specifically bind to" a specific target (such as TfR), or "be specific for" a specific target (such as TfR) can be demonstrated, for example, by a molecule with a dissociation constant K D for the bound target, for example, of 10 -4 M or less, such as 10 -5 M, 10 - 6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 M. In some embodiments, the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody specifically binds to an epitope that is conserved among species (e.g., structurally conserved among species), such as conserved between non-human primate and human species (e.g., structurally conserved between non-human primate and human species) on TfR. In some embodiments, the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding antibody can bind only to human TfR.

[0783] The term "variable region" or "variable domain" refers to the domain in an antibody heavy or light chain that is derived from germline variable (V), diversity (D), or joining (J) genes (and not from the constant (Cμ and Cδ) gene segments) and confers the specificity of the antibody for binding to an antigen. Typically, an antibody variable region includes four conserved "framework" regions interspersed with three hypervariable "complementary determining regions".

[0784] The terms "antigen-binding portion" and "antigen-binding fragment" are used interchangeably herein and refer to one or more fragments of an antibody that retain the ability to specifically bind an antigen through its variable regions. Examples of antigen-binding fragments include, but are not limited to, Fab fragments (monovalent fragments consisting of VL, VH, CL, and CH1 domains), F(ab') 2 fragments (bivalent fragments containing two Fab fragments linked by a disulfide bridge in the hinge region), single-chain Fv (scFv), disulfide-linked Fv (dsFv), complementary determining regions (CDRs), VL (variable light chain), and VH (variable heavy chain).

[0785] The following examples are intended to be non-limiting.

[0786] Example 1. Effect of peripheral administration of ETV:IDS on brain GAG and lysosomal lipids in IDS KO×TfR muhu mice

[0787] As described below, the effect of peripheral administration of ETV:IDS on brain GAG and lysosomal lipids in IDS KO×TfR muhu mice was studied.

[0788] Materials and Methods

[0789] Animal Care

[0790] Mice were housed on a 12-hour light / dark cycle and had free access to water and standard rodent chow ( #25502, irradiated).

[0791] Mouse Strains

[0792] Previously described IDS KO mice on a B6N background were obtained from The Jackson Laboratories (JAX strain 024744). The development and characterization of the TfR mu / hu KI mouse line with the human TfR apical domain knocked into the mouse receptor has been previously described (U.S. Patent No. 10,143,187). TfR mu / hu male mice were mated with female IDS heterozygous mice to generate IDS KO×TfR mu / hu mice. All mice used in this study were male.

[0793] Administration and tissue collection

[0794] Intravenously inject idursulfase (14.2 mg / kg body weight), or ETV:IDS (40 mg / kg body weight) into 2-month-old IDS KO×TfR mu / hu mice once a week for 4 weeks (n = 8). Intravenously inject saline into 2-month-old littermate TfR mu / hu mice once a week for 4 weeks (n = 5), which serves as a control. For the 7-day cohort, animals are sacrificed 7 days after the first administration. For the 28-day cohort, animals are sacrificed 7 days after the fourth administration of the weekly dosing.

[0795] To collect the final samples, animals are deeply anesthetized by intraperitoneal (i.p.) injection of 2.5% Avertin. To collect CSF, a sagittal incision is made at the posterior part of the animal's skull, the subcutaneous tissue and muscle are separated to expose the cisterna magna, and a pre-pulled glass capillary is used to puncture the cisterna magna to collect CSF. The CSF is transferred to a low-protein LoBind Eppendorf tube and centrifuged at 12,700 rpm for 10 minutes at 4°C. The CSF is transferred to a new tube and snap-frozen on dry ice. By measuring the absorbance of the sample at 420 nm, it can be confirmed that there is no blood contamination in the mouse CSF. Blood is collected by cardiac puncture to collect serum. To collect serum, the blood is allowed to clot at room temperature for at least 30 minutes. Then the tube is centrifuged at 12,700 rpm for 7 minutes at 4°C. The serum is transferred to a new tube and snap-frozen on dry ice. The animals are perfused transcardially with ice-cold PBS using a peristaltic pump (Gilson Minipuls Evolution). The brain and liver are dissected and snap-frozen on dry ice.

[0796] Tissue preparation: GAG extraction and processing

[0797] Brain and liver tissue samples and CSF are collected as described above. The brain and liver tissues are homogenized using a TissueLyser from Qiagen. Then the tissue homogenate is transferred to a 96-well deep plate and sonicated using a 96-tip sonicator (Q Sonica). A BCA protein assay is performed to quantify the total protein, and 20 μg of liver and 100 μg of brain from each sample are used for LC-MS / MS sample preparation. For CSF, 3 μL of each sample is used. Briefly, heparan sulfate- and dermatan sulfate-derived disaccharides are generated by digesting the tissue homogenate using a combination of heparinases I, II, III, and chondroitinase B. The digest is mixed with acetonitrile and then subjected to LC-MS / MS as described below.

[0798] LCMS determination of GAG

[0799] GAG quantification was performed by coupling liquid chromatography (Shimadzu Nexera X2 system, Shimadzu Scientific Instrument, Columbia, MD, USA) with electrospray mass spectrometry (Sciex QTRAP 6500+, Sciex, Framingham, MA, USA). For each analysis, the sample was injected into an ACQUITY UPLC BEH Amide 1.7 mm 2.1×150 mm column (Waters) at a flow rate of 0.55 mL / min, and the column temperature was 55 °C. Mobile phases A and B consisted of water containing 10 mM ammonium formate and 0.1% formic acid and acetonitrile containing 0.1% formic acid, respectively. The gradient program was as follows: 80% B from 0.0 - 0.5 min; 80% B to 50% B from 0.5 - 3.5 min; 50% B to 80% B from 3.5 - 4.0 min; held at 80% B from 4.0 - 4.5 min. Electrospray ionization was carried out in the negative ion mode with the following settings: curtain gas of 25; collision gas set to medium; ion spray voltage of -4500; temperature at 600 °C; ion source gas 1 at 50; ion source gas 2 at 60. Data acquisition was performed in multiple reaction monitoring mode (MRM) using Analyst 1.6.3 (Sciex), with a dwell time of 50 (milliseconds) for each substance. The collision energy (CE) was set to -30; the declustering potential (DP) was -80; the entrance potential (EP) was -10; the collision cell exit potential (CXP) was -10. GAG was detected as [M-H]- using the following MRM transitions: D0A0 at m / z 378.1>87.0; D0S0 at m / z 416.1>138.0; D0a4 at m / z 458.1>300.0; D4UA-2S-GlcNCOEt-6S (Iduron Ltd, Manchester, UK) at m / z 472.0 (source fragment ion)>97.0 was used as an internal standard. Individual disaccharide species were identified based on the retention time and MRM transitions of individual disaccharide species using commercially available reference standards (Iduron Ltd). GAG was quantified by comparing the peak area ratios of D0A0, D0S0, and D0a4 to the internal standard using MultiQuant 3.0.2 (Sciex). The reported GAG amount was normalized to the total protein level as measured by the BCA assay (Pierce).

[0800] Tissue preparation: Lipids were extracted from brain tissue

[0801] Transfer the frozen brain tissue (20 ± 2 mg) into a 2 mL Safe-Lock Eppendorf tube (Eppendorf catalog number 022600044) with dry ice and place it in dry ice containing 5 mm stainless steel beads (QIAGEN catalog number 69989) and 400 μL of MS-grade methanol with internal standard. Homogenize the tissue using a Tissuelyser at 25 Hz (in a cold room) for 30 seconds. Then centrifuge the sample at 21,000 × g for 20 minutes at 4 °C. Transfer the methanol supernatant to a new eppendorf vial and place it at -20 °C for 1 hour to allow further precipitation of proteins. Then centrifuge the sample at 21,000 × g for 10 minutes at 4 °C. Transfer 200 μL of the methanol supernatant to an LCMS 96-well plate and dry it under nitrogen, then resuspend it in 100 μL of ACN / IPA / H2O (92.5 / 5 / 2.5) with 5 mM ammonium formate and 0.5% formic acid for GlcCer analysis. Transfer the remaining supernatant to a separate LCMS 96-well plate without disturbing the pellet to analyze BMP and ganglioside species. Samples can be run directly on the LCMS or stored at -80 °C.

[0802] LCMS Determination of BMP and Gangliosides

[0803] BMP and ganglioside analysis was performed by combining liquid chromatography (Shimadzu Nexera X2 system, Shimadzu Scientific Instrument, Columbia, MD, USA) with electrospray mass spectrometry (Sciex QTRAP 6500+, Sciex, Framingham, MA, USA). For each analysis, 5 μL of sample was injected onto a BEH C18 1.7 μm 2.1×100 mm column (Waters Corporation, Milford, Massachusetts, USA) at a flow rate of 0.25 mL / min at 55 °C. Mobile phase A consisted of 60:40 acetonitrile / water (v / v) containing 10 mM ammonium acetate, and mobile phase B consisted of 90:10 isopropanol / acetonitrile (v / v) containing 10 mM ammonium acetate. The gradient program was as follows: 0.0 - 0.01 min, 45% B to 99% B; 0.1 - 3.0 min, 99% B; 3.0 - 3.01 min, to 45% B; and 3.01 - 3.50 min, at 45% B. Electrospray ionization was performed in the negative ion mode with the following settings: curtain gas was 30; collision gas was set to medium; ion spray voltage was -4500; temperature was 600 °C; ion source gas 1 was 50; ion source gas 2 was 60. Data acquisition was performed in multiple reaction monitoring mode (MRM) using Analyst 1.6.3 (Sciex) with the following parameters: dwell time (milliseconds) for each substance reported in Table A, collision energy (CE) of -50, declustering potential (DP) of -80; entrance potential (EP) of -10; and collision cell exit (CXP) potential of -15. BMP and ganglioside substances were quantified using the non-endogenous internal standards BMP di14:0 and GM3 (d36:1(d5)). Quantification was performed using MultiQuant 3.02 (Sciex). BMP and ganglioside concentrations were normalized relative to the total protein amount, tissue weight, or volume. Protein concentration was measured using the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL, USA).

[0804] Table A. Acquisition parameter information for BMP and ganglioside determination.

[0805]

[0806]

[0807]

[0808]

[0809] LCMS determination of GlcCer and GalCer

[0810] Glucosylceramide and galactosylceramide analyses were performed by coupling liquid chromatography (Shimadzu Nexera X2 system, Shimadzu Scientific Instrument, Columbia, MD, USA) with electrospray mass spectrometry (Sciex QTRAP 6500 + Sciex, Framingham, MA, USA). For each analysis, 10 μL of sample was injected onto a HALO HILIC 2.0 μm 3.0 × 150 mm column (Advanced Materials Technology, PN 91813 - 701) at a flow rate of 0.45 mL / min at 45 °C. Mobile phase A consisted of 92.5 / 5 / 2.5 ACN / IPA / H2O containing 5 mM ammonium formate and 0.5% formic acid. Mobile phase B consisted of 92.5 / 5 / 2.5 H2O / IPA / ACN containing 5 mM ammonium formate and 0.5% formic acid. The gradient program was as follows: 0.0 - 3.1 min, 100% B; 3.2 min, 95% B; 5.7 min, 85% B; held to 7.1 min, 85% B; decreased to 0% B at 7.25 min and held to 8.75 min; increased back to 100% at 10.65 min and held to 11 min. Electrospray ionization was carried out in positive ion mode with the following settings: curtain gas was 25; collision gas was set to medium; ion spray voltage was 5500; temperature was 350 °C; ion source gas 1 was 55; ion source gas 2 was 60. Data acquisition was performed in multiple reaction monitoring mode (MRM) using Analyst 1.6 (Sciex) with the following parameters: retention time (milliseconds) and collision energy (CE) for each substance reported in Table B; declustering potential (DP) was 45; entrance potential (EP) was 10; and collision cell exit potential (CXP) was 12.5. Lipids were quantified using a mixture of isotopically labeled internal standards as reported in Table B. Glucosylceramide and galactosylceramide were identified based on their retention times and the MRM characteristics of commercially available reference standards (Avanti Polar Lipids, Birmingham, AL, USA). Quantification was performed using MultiQuant 3.02 (Sciex). Metabolites were normalized relative to total protein amount, tissue weight, or volume.

[0811] Table B: Acquisition parameter information for GlcCer and GalCer determination.

[0812]

[0813]

[0814]

[0815] Mass spectrometry analysis of eicosanoids

[0816] Eicosanoid analysis was performed by combining liquid chromatography (Shimadzu Nexera X2 system, Shimadzu Scientific Instrument, Columbia, MD, USA) with electrospray mass spectrometry (Sciex QTRAP 6500+, Sciex, Framingham, MA, USA). For each analysis, 5 μL of the sample was injected onto a BEH C18 1.7 μm 2.1×100 mm column (Waters Corporation, Milford, Massachusetts, USA) at a flow rate of 0.6 mL / min at 40 °C. The mobile phase was composed as follows: A = water + 0.1% acetic acid, and B = 90:10 acetonitrile / isopropanol (v / v). The gradient program was as follows: 0.0 - 1.0 min, 25% B; 1.0 - 8.5 min to 95% B; 8.50 - 8.51 min, 95% B; 8.51 - 10.00 min, 25% B. Electrospray ionization was performed in the negative ion mode with the following settings: curtain gas was 30; collision gas was set to medium; ion spray voltage was -4500; temperature was 600 °C; ion source gas 1 was 50; ion source gas 2 was 60. Data acquisition was performed in multiple reaction monitoring (MRM) mode using Analyst 1.6.3 (Sciex) with the following parameters: retention time (milliseconds), collision energy (CE), and declustering potential (DP) for each substance reported in Table C; entrance potential (EP) was -10; and collision cell exit potential (CXP) was -12. Eicosanoids were quantified using a mixture of non-endogenous deuterated internal standards as reported in Table C. Eicosanoids were identified based on their retention times and the MRM characteristics of commercially available reference standards (Avanti Polar Lipids, Birmingham, AL, USA). Quantification was performed using MultiQuant 3.02 (Sciex) and Skyline. Metabolites were normalized relative to the total protein amount, tissue weight, or volume. Protein concentration was measured using the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL, USA).

[0817] Table C. Acquisition parameter information for eicosanoid determination.

[0818]

[0819]

[0820] Lipidomics analysis

[0821] Lipid analysis was performed by combining liquid chromatography (Shimadzu Nexera X2 system, Shimadzu Scientific Instrument, Columbia, MD, USA) with electrospray mass spectrometry (QTRAP 6500+, Sciex, Framingham, MA, USA). For each analysis, 5 μL of the sample was injected onto a BEH C18 1.7 μm 2.1×100 mm column (Waters Corporation, Milford, Massachusetts, USA) at a flow rate of 0.25 mL / min at 55 °C. For positive ionization mode, mobile phase A consisted of 60:40 acetonitrile / water (v / v) containing 10 mM ammonium formate + 0.1% formic acid; mobile phase B consisted of 90:10 isopropanol / acetonitrile (v / v) containing 10 mM ammonium formate + 0.1% formic acid. For negative ionization mode, mobile phase A consisted of 60:40 acetonitrile / water (v / v) containing 10 mM ammonium acetate; mobile phase B consisted of 90:10 isopropanol / acetonitrile (v / v) containing 10 mM ammonium acetate. The gradient program was as follows: 0.0 - 8.0 minutes, 45% B to 99% B; 8.0 - 9.0 minutes, 99% B; 9.0 - 9.1 minutes, to 45% B; and 9.1 - 10.0 minutes, 45% B. Electrospray ionization was performed in positive or negative ion mode using the following settings: curtain gas was 30; collision gas was set to medium; ion spray voltage was 5500 (positive mode) or 4500 (negative mode); temperature was 250 °C (positive mode) or 600 °C (negative mode); ion source gas 1 was 50; ion source gas 2 was 60. Data acquisition was performed in multiple reaction monitoring (MRM) mode using Analyst 1.6.3 (Sciex) with the following parameters: dwell time (milliseconds) and collision energy (CE) for each substance reported in Table D (negative mode) or Table E (positive mode); declustering potential (DP) was 80 (positive mode) and -80 (negative mode); entrance potential (EP) was 10 (positive mode) or -10 (negative mode); and collision cell exit potential (CXP) was 12.5 (positive mode) or -12.5 (negative mode). Lipids were quantified using a mixture of non-endogenous internal standards reported in Tables D and E. Lipids were identified based on their retention times and the MRM characteristics of commercially available reference standards (Avanti Polar Lipids, Birmingham, AL, USA). Quantification was performed using MultiQuant 3.02 (Sciex). Metabolites were normalized relative to the total protein amount or cell number.

[0822] Table D. Acquisition parameter information for lipidomics assays in negative mode.

[0823]

[0824]

[0825]

[0826]

[0827] Table E. Acquisition parameter information for lipidomics determination in positive mode.

[0828]

[0829]

[0830]

[0831]

[0832]

[0833]

[0834]

[0835]

[0836] ETV:IDS brain tissue homogenization and Trem2 analysis of ETV:IDS brain tissue and CSF

[0837] Homogenize 50 mg of tissue in 500 μL of 1X CST buffer (Cell Signaling Technology 9803S) made with complete protease inhibitor (Roche #04693132001) and PhosStop (Roche 04906837001) at 30 Hz for 2 rounds of 3 minutes using a Qiagen TissueLyzer II (Catalog number / ID: 85300). Incubate the homogenate on ice for 20 minutes and spin at 21,100 g at 4 °C for 30 minutes. Subsequently, transfer the lysate to a clean 96-well deep plate and perform BCA to quantify the total protein amount. Store the samples at -80 °C until use in the assay.

[0838] For Trem2 analysis in brain tissue and soluble Trem2 (sTrem2) analysis in CSF, MSD GOLD 96w multi-spot streptavidin plates (MSD L45SA) were prepared for Trem2 assays by overnight coating with 1 μg / mL biotinylated sheep anti-mouse antibody (R&D Systems BAF1729) at 4 °C. The next day, the MSD plates were washed with tris-buffered saline containing triton (TBST) and blocked with TBST containing 3% bovine serum albumin for two hours with shaking at 600 rpm. The MSD plates were washed again with TBST, and brain lysates were diluted 5-fold in the blocking solution and then added to the MSD plates and incubated for 1 hour at 600 rpm. After the next TBST wash, sulfonated sheep anti-mouse antibody (R&D Systems AF1729) was added to the plates and incubated again for 1 hour at 600 rpm, followed by a final wash and then addition of 2X MSD read buffer diluted in water. The plates were then read using an MSD Meso Sector S600. The Trem2 signal was normalized relative to the protein concentration and graphed using GraphPad Prism.

[0839] Abbreviations

[0840] BMP = bis(monoacylglycerol)phosphate; ETV:IDS = enzyme transporter vehicle: iduronate 2-sulfatase; GlcCer = glucosylceramide; GalCer = galactosylceramide; IDS = iduronate 2-sulfatase; KI = knock-in; KO = knock-out; TfR mu / hu = chimeric human / mouse transferrin receptor.

[0841] Results

[0842] To determine whether the strong GAG reduction observed in the brain translates into correction of downstream disease-related pathologies, the ability of ETV:IDS to correct secondary lysosomal storage was evaluated. Active equivalent doses of ETV:IDS or idursulfase were intravenously administered once weekly for four weeks to IDS KO; TfR mu / hu KI mice, and the levels of a panel of lysosomal lipids, including gangliosides, glucosylceramide, and bis(monoacylglycerol)phosphate, were measured using liquid chromatography-tandem mass spectrometry (LCMS). Compared to wild-type controls, in IDS KO; TfR mu / huA large accumulation of lysosomal lipids was observed in the brains of KI mice. After administering 40 mg / kg once a week for four weeks, ETV:IDS was highly effective in reducing lysosomal lipids in the brain, completely reducing the levels of these lipids to those seen in wild-type mice. However, treatment with an enzymatically equivalent dose of idursulfase did not reduce the levels of these lysosomal lipids in the brain. Together, these data demonstrate that ETV:IDS can effectively correct secondary lysosomal storage in addition to its proximal effect on GAG accumulation.

[0843] Specifically, Figures 5A - 5B showed that peripheral administration of ETV:IDS (4-week treatment) corrected ([[]] Figure 5A ) brain and ([[]] Figure 5B ) CSF GAG accumulation in IDS KO mice. Compared with a 14% reduction with an enzymatically equivalent dose of Elaprase, brain GAG was reduced by 58% when ETV:IDS was peripherally administered. In addition, compared with a 27% reduction with an enzymatically equivalent dose of Elaprase, a 66% reduction in CSF GAG was observed when ETV:IDS was peripherally administered (one outlier in the ETV:IDS group was omitted). Figures 14A - 14B showed a reduction in individual GAG species (D00S0, D00A0, D00a4) in the brain and CSF. In a follow-up study, at an ETV:IDS dose of 40 mg / kg, CSF GAG was reduced three-fold compared to WT.

[0844] Figure 6 showed that peripheral administration of ETV:IDS (4-week treatment) corrected the accumulation of lysosomal lipids (gangliosides) in the brains of IDS KO mice.

[0845] Figures 7A - 7B showed that peripheral administration of ETV:IDS (4-week treatment) corrected the accumulation of lysosomal lipids (GlcCer) in the brains of IDS KO mice. Correction of brain GlcCer accumulation was observed with peripheral administration of ETV:IDS, while no change in galactosylceramide (GalCer) was observed.

[0846] Figure 8 showed that peripheral administration of ETV:IDS (4-week treatment) corrected the accumulation of lysosomal lipids (BMP) in the brains of IDS KO mice.

[0847] Figure 9 showed a heat map of lipid levels in the brains of IDS KO mice treated with vehicle, ETV:IDS, or Elaprase (idursulfase). This heat map was generated using the modified data from Table 2. Specifically, the data in Table 2 were modified using a cut-off value to convert values between 0.9 and 1.1 to 1 to show differences greater than 10%.

[0848] Figure 10 Peripheral administration of ETV:IDS corrected TREM2 accumulation in the brains of IDS KO mice.

[0849] Figure 11 Shows CSF sTrem2 levels relative to TfR mu / hu KI mice were increased in the IDS KO; TfR mu / hu KI mouse cohort. Treatment with ETV:IDS reduced IDS KO; TfR mu / hu KI mouse CSF sTrem2 level accumulation.

[0850] Table 1 summarizes the GlcCer species analyzed, with levels expressed as multiples of WT.

[0851] Table 2 provides data on lipid levels in the brains of IDS KO mice treated with vehicle, ETV:IDS, or Elaprase (fold change relative to WT).

[0852] Example 2. Measuring Brain GAG, Lysosomal Lipids, and Neurofilament Light Chain (Nf-L) in 3-, 6-, and 9-Month-Old IDS KO Mice

[0853] As described below, brain GAG, lysosomal lipid, and neurofilament light chain (Nf-L) levels were studied in 3-, 6-, and 9-month-old WT and IDS KO mice.

[0854] Materials and Methods

[0855] As described herein (Example 1), animals used in this study were cared for. Tissues were sampled, and lipid and GAG levels were measured using the methods described herein (Example 1). Nf-L levels were measured as described below.

[0856] Methods for CSF and Serum Analysis of Nf-L

[0857] Using the Quanterix Simoa Neurofilament Light Chain (NF-L) Sample Diluent (Quanterix 102252), cerebrospinal fluid (CSF) was diluted 100-fold and serum was diluted 4-fold, and then added to the Simoa 96-well microplate (Quanterix 101457). According to the instructions of the Simoa NF-Light Advantage Kit (Quanterix 1031086), the NF-light assay was performed using the Simoa detection reagent and bead reagent (Quanterix 103159 and 102246, respectively). After incubating the sample with the detection reagent and bead reagent at 30 °C and 800 rpm for 30 minutes, the sample plate was washed with Simoa Wash Buffer A (Quanterix 103078) on the Simoa microplate washer according to the Quanterix two-step protocol. Subsequently, the SBG reagent (Quanterix 102250) was added, and the sample was incubated at 30 °C and 800 rpm for 10 minutes. The two-step washer protocol was continued, and the sample beads were resuspended twice in Simoa Wash Buffer B (Quanterix 103079), and then the buffer was finally aspirated. The sample NF-L level was measured on the Quanterix SR-X instrument using the NF-Light analysis protocol and interpolated according to the calibration curve provided by the Quanterix assay kit.

[0858] Results

[0859] Measurement of the accumulation of brain HS / DS (GAG) in IDS KO mice relative to age-matched WT controls at 3, 6, and 9 months of age ( Figure 1 ). In IDS KO mice at all tested ages, GAG accumulation was significantly higher.

[0860] The lysosomal lipid accumulation (GM1, GM2, GM3, BMP, GlcCer, and GD3) in the brains of IDS KO mice relative to age-matched WT controls was also measured ( Figures 2A - 2D ). GM1 (d36:1) showed similar levels in IDS KO and WT mice ( Figure 2A and 2B ), while the levels of GM2 (36:1), GM3 (36:1), BMP (36:2), GlcCer (34:1), and GD (39:1) showed a 1.7- to 5.5-fold increase compared to WT animals ( Figures 2A - 2D ). Similar to GD3 (39:1), GD3 (36:1) also increased in IDS KO (on average 2-fold).

[0861] Elevated BMP levels were also observed in the sera of IDS KO mice relative to age-matched controls. Specifically, in 9-month-old serum samples collected from IDS KO mice, BMP(36:2) and BMP(44:12) were particularly increased compared to WT controls( Figure 3 ).

[0862] Elevated lysosomal lipid levels (Gd1a / b, GM3, BMP, and GlcCer) were observed in the CSF of 9-month-old IDS KO mice relative to WT age-matched controls( Figure 4 ).

[0863] Nf-L is a useful marker of neurodegeneration (Norgren et al. 2003. Brain Research 987(1):25-31), but has not previously been associated with Hunter syndrome / MPSII. Elevated Nf-L concentrations were observed in the sera and CSF of 9-month-old IDS KO mice relative to an age-matched wild-type mouse cohort( Figure 26A , Figure 26B ). The relative difference in Nf-L levels in the CSF increased with the age of the mouse cohort, with the largest difference in Nf-L levels observed in the 9-month-old mouse cohort group( Figure 26C ). These results suggest that Nf-L may also be used as a disease-related and treatment-responsive biomarker in a mouse model of Hunter syndrome.

[0864] Example 3. Peripheral administration of ETV:IDS effects on GAG and lysosomal lipids in IDS KO×TfR muhu mice

[0865] Examine the effects of varying doses of ETV:IDS on GAG and lysosomal lipids in IDS KO×TfR muhu mice.

[0866] Materials and methods

[0867] Animal care

[0868] Mice were housed on a 12-hour light / dark cycle and had free access to water and a standard rodent diet( #25502, irradiated).

[0869] Mouse strains

[0870] The IDS KO×TfR mu / hu mice used in this study were described in Example 1 above. All mice used in this study were male.

[0871] Administration and sample collection

[0872] Intravenously inject saline, idursulfase (14.2 mg / kg body weight), or ETV:IDS (3, 10, 20, or 40 mg / kg body weight) into IDS KO×TfR mice at 2 - 3 months of age once a week for 4 weeks (n = 5 - 8). Intravenously inject saline into littermate TfR mice at 2 - 3 months of age once a week for 4 weeks (n = 5), which serves as a control. Sacrifice the animals 7 days after the last administration of the 4 - week drug treatment. mu / hu mu / hu

[0873] To collect the final samples, deeply anesthetize the animals by intraperitoneal (i.p.) injection of 2.5% Avertin. To collect CSF, make a sagittal incision at the posterior part of the animal's skull, separate the subcutaneous tissue and muscle to expose the cisterna magna, and use a pre - pulled glass capillary to puncture the cisterna magna to collect CSF. Transfer the CSF to a low - protein LoBind Eppendorf tube and centrifuge at 12,700 rpm for 10 minutes at 4°C. Transfer the CSF to a new tube and quickly freeze it on dry ice. By measuring the absorbance of the sample at 420 nm, it can be confirmed that there is no blood contamination in the mouse CSF. Collect blood by cardiac puncture to collect serum. To collect serum, allow the blood to clot at room temperature for at least 30 minutes. Then centrifuge the tube at 12,700 rpm for 7 minutes at 4°C. Transfer the serum to a new tube and quickly freeze it on dry ice. Perfuse the animals transcardially with ice - cold PBS using a peristaltic pump (Gilson Minipuls Evolution). Dissect the brain and quickly freeze it on dry ice.

[0874] Tissue preparation and LCMS determination

[0875] Perform tissue preparation and LCMS determination using a method similar to that described in Example 1.

[0876] Results

[0877] For mice receiving ETV:IDS, a dose - dependent decrease in serum, brain, and CSF GAG was observed ( Figures 12A - 12C ). Notably, serum GAG was corrected to wild - type levels.

[0878] Importantly, even at the lowest ETV:IDS dose tested (3 mg / kg), brain lysosomal lipids (GM3, GlcCer, and BMP) were significantly reduced ( Figures 13A - 13C ). Since lipid accumulation in IDS - deficient mice is thought to be a functional consequence of GAG accumulation, these results may indicate that even with elevated GAG levels relative to WT mice, functional restoration of lysosomal function in the brain can be achieved.

[0879] ​​Example 4. Sorting Specific CNS Cell Types from Brain Tissue

[0880] A protocol was developed to isolate enriched populations of neurons, astrocytes, and microglia from brain tissue. The enriched populations were then used to study the effects of administering ETV:IDS in IDS KO×TfR mu / hu mice (Example 5).

[0881] Materials and Methods

[0882] Animal Care

[0883] Mice were housed on a 12-hour light / dark cycle and had free access to water and standard rodent chow ( #25502, irradiated).

[0884] CNS Cell Type Isolation

[0885] To prepare a single-cell suspension for sorting CNS cells, mice were perfused with PBS, the brains were dissected, and processed into a single-cell suspension using an Adult Brain Dissociation Kit (Miltenyi Biotec 130-107-677) according to the manufacturer's protocol. Cells were Fc-blocked (Biolegend #101320, 1:100) and stained with Fixable Viability Stain BV510 (BD Biosciences #564406, 1:100) (to exclude dead cells), CD11b-BV421 (BD Biosciences 562605, 1:100), CD31-PerCP Cy5.5 (BD Biosciences #562861, 1:100), O1-488 (Thermo / eBio #14-6506-82, 1:37.5), Thy1-PE (R&D #FAB7335P, 1:100), and EAAT2-633 (Alomone #AGC-022-FR, 1:50) for flow cytometry analysis. Cells were washed with PBS / 1% BSA and filtered through a 100 μm filter, then CD11b+ microglia, EAAT2+ astrocytes, and Thy1+ neurons were sorted on a FACS Aria III (BD Biosciences) with a 100 μm nozzle. To obtain pure populations of astrocytes, microglia, and neurons, negative gates were set to remove O1+ and CD31+ cells, which are mainly oligodendrocytes and endothelial cells, respectively. The sorted cell pellets were either pelleted or directly collected into lysis buffer to prepare for downstream analysis, including qRT-PCR, RNAseq, or glycomics, as described in the relevant methods disclosed herein. Cell numbers were used to calculate pg GAG / cell.

[0886] Figure 15A Schematic of the CNS cell sorting protocol for separating pure neuronal, astrocyte, and microglial cell populations and downstream endpoints analyzed for the administration of ETV:IDS to mice (Example 5). Figure 15B Flowchart of the gating protocol for separating enriched cell populations. Figure 15C Includes representative FACS gating for the sorting procedure. Starting from the upper left to the lower right: forward (FSC) and side (SSC) scatter determine cells in debris; live cell main gating; confirmation of exclusion of CD31-positive endothelial cells; EAAT2-positive astrocytes from CD11b microglial sub-gating; Thy1-positive neurons from EAAT2-positive astrocyte sub-gating; and finally removal of O1 oligodendrocytes in the CD11b microglial, EAAT2 astrocyte, and Thy1 neuronal cell populations determines the final sorting criteria.

[0887] RNAseq and qPCR analysis of gene expression of isolated CNS cell types

[0888] To validate the sorting method, the expression of neuronal, astrocytic, and microglial genes in the sorted cell populations was analyzed by RNAseq and qPCR. Live cells were directly sorted in 350 μL of RLT-plus buffer (Qiagen, Hilden, Germany) containing 1:100 β-mercaptoethanol. RNA was extracted using the RNeasy Plus Micro Kit (Qiagen, 74034) and resuspended in 14 μL of nuclease-free water. The quantity and quality of RNA were evaluated using an RNA 6000 Pico Chip (Agilent 5067-1513) on a 2100 Bioanalyzer (Agilent). For qPCR validation, 1-2 μL of RNA was transcribed into cDNA using SuperScript IV (Invitrogen). Gene expression of target genes was evaluated on a QuantStudio6Flex (Applied Biosystems) using Taqman probes and normalized relative to Gapdh. For QuantSeq library preparation, the manufacturer-defined 'low input' protocol was followed using the Illumina QuantSeq 3' mRNAseq Library Prep Kit FWD (Lexogen). RNA was processed using the UMI second-strand synthesis module to identify and remove PCR duplicate fragments. Barcoded samples were quantified using the Illumina NEBNext Library Quantification Kit (NEB, E7630S). All samples were pooled into a sequencing library at equimolar ratios and quantified on a Bioanalyzer (Agilent, 5067-4626) with a high-sensitivity DNA chip. 50-bp single-end reads were generated on an Illumina HiSeq 4000 lane at the UCSF Center for Advanced Technology.

[0889] For RNAseq raw data processing, UMI was extracted from the raw sequencing reads using umi2index (Lexogen), and the sequencing adapters were trimmed with skewer (Jiang et al. 2014. BMC Bioinformatics 15:182). The reads were aligned to the mouse genome version GRCm38_p6. The STAR index (version 2.5.3a) was built using the –sjdbOverhang=50 argument (Dobin et al. 2013. Bioinformatics 29:15-21). The splice junctions of the Gencode gene model (release M17) were provided through the –sjdbGTFfile argument. The STAR alignment was generated using the following parameters: -outFilterType BySJout, -quantMode TranscriptomeSAM, -outFilterIntronMotifs RemoveNoncanonicalUnannotated, -outSAMstrandField intronMotif, -outSAMattributes NH HI AS nM MD XS, and -outSAMunmapped Within. The alignment was obtained using the following parameters: -readFilesCommand zcat -outFilterType BySJout -outFilterMultimapNmax 20 -alignSJoverhangMin 8 -alignSJDBoverhangMin 1

[0890] -outFilterMismatchNmax 999 -outFilterMismatchNoverLmax 0.6

[0891] -alignIntronMin 20 -alignIntronMax 1000000 -alignMatesGapMax 1000000

[0892] -quantMode GeneCounts - outSAMunmapped Within - outSAMattributes NH HI AS nM MD XS - outSAMstrandField intronMotif - outSAMtype BAM SortedByCoordinate - outBAMcompression 6. The alignment mapping to the same genomic location sharing the same UMI was collapsed using the collapse_UMI_bam tool (Lexogen). Gene - level counts were obtained using featureCounts from the Subread package (version 1.6.2) (Liao et al. 2013. Bioinformatics 30:923 - 930). Gene symbol and biotype information were extracted from the Gencode GTF file.

[0893] All RNA - seq expression analyses were performed using R (R Core Team 2018; version 3.2), with the voom analysis framework from the limma package (Ritchie et al. 2015. Nucleic Acids Research 43:e47) (Law et al. 2014. Genome biology 15:R29). Gene expression profiles were normalized by TMM (Robinson and Oshlack. 2010. Genome Biology 11:R25) and low - abundance genes were identified and removed prior to downstream analysis. Low - abundance genes were defined as genes with an expression of no more than 10 (ten) counts per million (CPM) in at least four samples.

[0894] For the principal component analysis to determine which variables explain the major differences between samples, the log - transformed CPM expression values from the top 500 genes with the highest variance were used for principal component analysis. The projection of samples on the first two principal components is shown as Figure 16 shown. Principal components 1 and 2 accounted for 48% and 26% of the data variance, respectively.

[0895] Marker genes were identified for each cell type by combining the results of separate pairwise differential expression tests between itself and the other two cell types. The marker gene p-value for each gene was calculated by combining the nominal p-values from two "outgroup" differential expression tests using the Simes method (Simes, R.J. 1986. Biometrika 73:751-754). The false discovery rate (FDR) was calculated from the combined p-values using the Benjamini-Hochberg method (Benjamini and Hochberg. 1995. Journal of the Royal Statistical Society. Series B (Methodological) 57:289-300). Genes were sorted by decreasing the average fold change in logarithm compared to the other two cell types, and the top 20 genes with FDR < 0.01 were used as marker genes for the cells. Separate pairwise differential expression tests were performed using limma / voom. To identify genes with strong expression enrichment in the target cell type relative to the rest of the cells, the treat framework of limma was used to test for statistical significance relative to a five-fold change threshold (Robinson and Oshlack, ibid).

[0896] Results

[0897] To confirm the enrichment and purity of the isolated cell populations, the gene expression profiles of each sorted population were analyzed using RNA-Seq and qRT-PCR and compared to known profiles ( Figure 16 , Figures 17A - 17C and Figure 25 ). Strong enrichment of the corresponding cell type-specific genes in the isolated populations was observed, as well as a reduction in the gene markers for endothelial cells and oligodendrocytes. This pooled data indicates that highly pure populations of neurons, astrocytes, and microglia were obtained.

[0898] Figure 17A Shows the expression of classical cell-specific markers identified in the literature in purified neurons, astrocytes, microglia, and input cell suspensions determined by RNA-Seq. The rows are the genomes specific to the cell types: (endo.) endothelial, (oligo.) oligodendrocyte; the expression value for each gene is depicted as the number of standard deviations from its mean (z-score); n = 4 mice.

[0899] Figure 17BThe expression of the top 20 enriched genes determined by an enrichment fold >5.0 and FDR <0.01 for each cell type is listed and shown. Heatmap expression values are plotted as gene-level z-scores. The input cells are single-cell suspensions from dissociated brains.

[0900] Figure 17C Including representative qRT-PCR data generated from isolated cell populations, confirming that these populations have been successfully enriched for neurons, astrocytes, and microglia. Gene categories are grouped on the x-axis: (Astro) astrocytes, (MG) microglia, (Neu) neurons, (Oligo) oligodendrocytes, and (Endo) endothelial cells; n = 5 mice. The graph shows mean ± SEM.

[0901] Figure 25 is a table of cell type-specific enriched gene sets; the information in the table corresponds to the Figure 17B heatmap. The expression of the top 20 genes determined by an enrichment fold >5.0 and FDR <0.01 for each cell type is listed in ascending order of p-value. The average fold change in logarithm (logFC.avg) is relative to two other "outgroup" populations, and the rightmost three columns show the average expression of genes within each cell population; n = 4 mice.

[0902] Example 5. Effects of peripherally administered ETV:IDS on GAG and lysosomal lipids in specific CNS cell types of IDS KO×TfR muhu mice

[0903] Examine the effects of varying doses of ETV:IDS on GAG and lysosomal lipids in specific CNS cell types of IDS KO×TfR muhu mice.

[0904] Materials and Methods

[0905] Animal Care

[0906] Mice were housed under a 12-hour light / dark cycle and had free access to water and standard rodent diet ( #25502, irradiated).

[0907] Mouse Strains

[0908] The TfR mu / hu mice and IDS KO×TfR mu / hu mice used in this study were described in Example 1 above. All mice used in this study were male.

[0909] Administration and Sample Collection

[0910] To 2- to 3-month-old IDS KO×TfRmu / hu Mice were intravenously injected with saline or ETV:IDS (40 mg / kg body weight) once a week for 4 weeks (n = 4 - 6 per treatment). For littermate TfR at 2 - 3 months of age mu / hu Mice were intravenously injected with saline once a week for 4 weeks (n = 4 - 6), which served as the control. Animals were sacrificed 7 days after the last administration of the 4 - week drug treatment.

[0911] To collect the final samples, animals were deeply anesthetized by intraperitoneal (i.p.) injection of 2.5% Avertin. To collect CSF, a sagittal incision was made at the posterior part of the animal's skull, and the subcutaneous tissue and muscle were separated to expose the cisterna magna. A pre - pulled glass capillary was used to puncture the cisterna magna to collect CSF. The CSF was transferred to a low - protein LoBind Eppendorf tube and centrifuged at 12,700 rpm for 10 minutes at 4°C. The CSF was transferred to a new tube and snap - frozen on dry ice. Absence of blood contamination in mouse CSF was confirmed by measuring the absorbance of the sample at 420 nm. Blood was collected by cardiac puncture to obtain serum. To collect serum, the blood was allowed to clot at room temperature for at least 30 minutes. Then the tube was centrifuged at 12,700 rpm for 7 minutes at 4°C. The serum was transferred to a new tube and snap - frozen on dry ice. The animals were perfused transcardially with ice - cold PBS using a peristaltic pump (Gilson Minipuls Evolution). The brain was dissected and snap - frozen on dry ice.

[0912] Isolation of CNS cell types

[0913] CNS cells were sorted as described to achieve pure populations of astrocytes, microglia, and neurons (Example 4). The sorted cell pellets were either pelleted or directly collected into lysis buffer and then processed for downstream analysis, including qRT - PCR, RNAseq, or glycomics, as described in the relevant methods. Cell numbers were used to calculate pg GAG / cell.

[0914] Cell lysate preparation and LCMS assays for measuring GAG, BMP, gangliosides, GlcCer, and GalCer were performed using a method similar to that described in Example 1.

[0915] Analysis of the distribution of ETV:IDS across CNS cell types

[0916] Live cells in sheath fluid (approx. 1.5 ml) were directly sorted into 150 μL of 5% CHAPS lysis buffer, with a final CHAPS concentration of 0.5%. Samples were concentrated using an Amicon Ultra 30KDa filter. Five (5) μL of sample or recombinant ETV:IDS series dilutions were assayed using an IgG (human) AlphaLISA detection kit (PerkinElmer #AL205C) according to the manufacturer's instructions and read on an EnVision TM plate reader. Sample concentrations were interpolated from a standard curve generated using ETV:IDS and normalized relative to the total cell input number.

[0917] Results

[0918] Evaluate the cell type-specific distribution and efficacy of ETV:IDS in the brains of IDS KO; TfR mu / hu KI mice.

[0919] GAG levels in enriched CNS cell populations were quantified by LC-MS / MS as described. Figure 18A Shown relative to TfR mu / hu KI controls, elevated GAG levels were observed in microglia, astrocytes, and neurons isolated from IDS KO; TfR mu / hu KI mice, demonstrating substrate accumulation in all three CNS cell types when IDS expression was knocked out (n = 3 - 5 mice per group). Forty mg / kg of ETV:IDS was administered intravenously to IDS KO; TfR mu / hu KI mice (n = 4 mice per group), and two hours after dosing, enzyme concentrations were evaluated in the entire CNS cell population. Figure 18B Shown is significant accumulation of ETV:IDS observed in neurons, astrocytes, and microglia, demonstrating that ETV:IDS is effectively distributed to the brain parenchyma and taken up by key CNS cell types. All data are shown as mean ± SEM; unpaired Student's t-test p ≤ 0.05*, 0.001***.

[0920] Next, 40 mg / kg of ETV:IDS was administered intravenously to IDS KO; TfR mu / hu KI mice once a week for four weeks, and LC-MS / MS was used to evaluate the ability of ETV:IDS to reduce GAG accumulation in all three CNS cell types. Figure 19After repeated administration, the ETV:IDS treatment reduced GAG levels in neurons, astrocytes, and microglia to levels comparable to those seen in wild-type mice. Data are shown as mean ± SEM; one-way ANOVA with Tukey's multiple comparison test; *p ≤ 0.05, **p ≤ 0.01. The data demonstrate that ETV:IDS is able to deliver IDS across the brain endothelium to brain cells and that this delivery is sufficient to exert efficacy in key CNS cell types.

[0921] The ETV:IDS treatment also reduced secondary accumulation of lysosomal lipids in the CNS cell types of interest (e.g., neurons, astrocytes, microglia), including gangliosides ( Figure 20 ), glucosylceramides ( Figure 21 ), and bis(monoacylglycerol)phosphate (BMP) ( Figure 22 ). These data suggest that treatment with ETV:IDS can correct secondary lysosomal dysfunction in addition to the primary GAG storage in CNS cell types.

[0922] Example 6. Spatial distribution of accumulated lipid species and correction of lipid accumulation by ETV:IDS

[0923] Examine the effects of varying doses of ETV:IDS on the spatial distribution of lysosomal lipids and microglial activation in IDS KO×TfR muhu mice.

[0924] Mouse strains and administration

[0925] The TfR mu / hu mice and IDS KO×TfR mu / hu mice used in this study were described in Example 1 above. All mice used in this study were male. Mice were administered saline, idursulfase, or ETV:IDS (40 mg / kg body weight) and sacrificed for tissue analysis as described in Example 1.

[0926] Mass spectrometry-based imaging of ganglioside levels

[0927] The brain tissue was snap-frozen on aluminum foil and then slowly lowered into liquid nitrogen for approximately 10 seconds. The frozen brain was stored at -80 °C until ready for use. Prior to sectioning, the brain was placed in a cryostat chamber to equilibrate the tissue to -20 °C. The brain was sectioned into 12-μm-thick slices on a cryostat (Leica Biosystems, Buffalo Grove, IL) and thaw-mounted onto glass slides (Delta Technologies, Loveland, CO) coated with indium tin oxide (ITO). Two brain levels were collected at approximately +0.72 mm and -1.82 mm from bregma. The plates with the slices designated for IMS were washed three times with cooled (ca. 4 °C) 50 mM ammonium formate and dried at room temperature before matrix application. Additional slices were obtained for H&E staining. After staining, digital micrographs were obtained using a slide scanner (Leica Biosystems, Buffalo Grove, IL). For matrix application, the plates were coated with 1,5-diaminonaphthalene (DAN) MALDI matrix by sublimation (Hankin et al. 2007. Journal of the American Society for Mass Spectrometry 18:1646-1652; Thomas et al. 2012. Analytical Chemistry 84:2048-2054). Briefly, 100 mg of recrystallized DAN was placed at the bottom of a glass sublimation apparatus (Chemglass Life Sciences, Vineland, NJ). The apparatus was placed on a metal heating block set to 130 °C, and DAN was sublimed onto the tissue surface at a pressure of less than 25 mTorr for 4 minutes. Approximately 1.8 mg of DAN was determined to be applied to each slide by weighing the slides before and after matrix application. The coated plates were then placed in a Petri dish, flushed with nitrogen, and stored at -80 °C for two days prior to MS analysis (Yang et al. 2019. International Journal of Mass Spectrometry 437:3-9).

[0928] For mass spectrometry imaging, the plates were allowed to equilibrate to room temperature before removal from the sealed Petri dishes. Brain sections were imaged on a Solarix 15T FT-ICR MS (Bruker Daltonics, Billerica, MA) equipped with a SmartBeam II 2 kHz frequency tripled Nd:YAG laser (355 nm). Images were acquired at a spatial resolution of 100 μm in negative ion mode. A smaller laser focus setting was used and a random walk within 100 μm pixels, with each pixel being the average of 1000 laser shots. The mass spectrometer was externally calibrated using a series of phosphorous clusters (Sládková et al 2009. Rapid Communications in Mass Spectrometry 23:3114-3118). Data were collected from m / z 600 - 3,000 with a time domain file size of 1M (FID length = 1.3631 s), resulting in a resolving power of 153,000 at m / z 1041. Images were generated using FlexImaging 3.0 (Bruker Daltonics, Billerica, MA). Gangliosides were identified by accurate mass, with mass accuracy typically better than 1 ppm.

[0929] Immunohistochemistry

[0930] Fresh frozen mouse brain tissue was coronally sectioned into 10-μm-thick slices using a Leica cryostat (Leica CM 1950). The slices were directly mounted onto Fisherbrand Superfrost Plus microscope slides and stored at -80 °C until processed for immunohistochemistry. The slices were rinsed 3 times for 5 minutes each in 1x PBS, then fixed in 4% paraformaldehyde for 15 minutes. The slices were then rinsed 3 times for 5 minutes each in 1x PBS, then incubated in blocking solution (1x PBS / 5% normal goat serum / 0.3% Triton X-100) for 1 hour at room temperature. The slices were then incubated in primary antibody (BioRad: rat anti-Cd68, 1:500) prepared in blocking solution for 2 hours at room temperature. The slices were rinsed 3 times for 5 minutes each in 1x PBS / 0.3% Triton X-100, then incubated in secondary antibody (Invitrogen: goat anti-rat Alexa Fluor 488, 1:500) and DAPI (Invitrogen Molecular Probes D1306: starting from 5 mg / mL stock solution, 1:10,000) prepared in blocking solution for 1 hour at room temperature in the dark. The slices were then rinsed 3 times for 5 minutes each in 1x PBS / 0.3% Triton X-100, quickly rinsed in 1X PBS, and then coverslipped with polyvinyl alcohol mounting medium with DABCO anti-fade (Sigma 10981). Fluorescent images were acquired using a Zeiss Axio Scan Z1 at 20x magnification. Each fluorophore was imaged separately using appropriate single-channel filter sets, and the exposure time for each fluorophore was the same in all tissue samples imaged. Individual images were then tiled and stitched using Zeiss Zen software, and shadow correction was performed.

[0931] Results

[0932] Mass spectrometry imaging (IMS) was performed to determine the spatial distribution of lipid accumulation in the brains of IDS KO;TfR mu / hu KI mice and to evaluate whether ETV:IDS administration was able to correct lysosomal lipid accumulation throughout the brain. MALDI MS images were obtained from coronal brain slices of wild-type and IDS KO;TfR mu / hu KI mice after administration of vehicle, idursulfase, or ETV:IDS once weekly for four weeks. Representative images of selected ganglioside species are shown in Figure 23As shown, the middle panel shows the signal distribution at m / z 1382.816, corresponding to GM2(d36:1), while the bottom panel shows the signal distribution at m / z 1179.738, corresponding to GM3(d36:1). The image depicts the relative intensity of each signal from 0 - 100%.

[0933] As Figure 23 shown, enrichment of several ganglioside species was observed in the brains of IDS KO; TfR mu / hu KI mice compared to wild - type controls. This is similar to the results observed in the analysis of homogenized tissues ( Figure 6 ). Ganglioside accumulation was region - specific and concentrated in the hypothalamus and amygdala brain regions. Administration of ETV:IDS once a week for four weeks reduced the accumulation of these ganglioside species throughout the brain regions, and the levels were evaluated to be the same as those seen in wild - type mice, while only a slight reduction in gangliosides was observed under idursulfase treatment. Collectively, these data suggest that ETV:IDS can correct lysosomal dysfunction downstream of GAG accumulation in the CNS.

[0934] Neuroinflammation represents a common feature of many neurodegenerative LSDs, and there has emerged a consensus that glial activation commonly reported in mouse models of MPS II disease as well as in MPS patients may contribute to progressive degeneration throughout the brain in MPS disorders. Two markers of microglial activity, namely CD68 and triggering receptor expressed on myeloid cells 2 (Trem2), were evaluated by immunohistochemical analysis of brain tissue sections or biochemical analysis of brain lysates, respectively. Figure 24 are representative images of immunofluorescence staining of DAPI and CD68 in the brains of TfR mu / hu KI and IDS KO; TfR mu / hu KI mice treated with vehicle, ETV:IDS, or idursulfase. The magnification is 20 - fold, and the images represent the hippocampus (top panel), cortex (middle panel), and striatum (bottom panel). Figure 10 shows the levels of Trem2 in the treated mice.

[0935] Compared to TfR mu / hu KI controls, the levels of both CD68 and Trem2 were elevated in the brains of IDS KO; TfR mu / hu KI mice ( Figure 10 , Figure 24 ). Administration of 40 mg / kg ETV:IDS once a week for four weeks effectively reduced the CD68 signal throughout the brain of IDS KO; TfR mu / hu KI mice ( Figure 21)。After repeated systemic administration of ETV:IDS, the levels of Trem2 in the brain were completely reduced to the levels seen in wild-type mice ( Figure 10 ). However, weekly administration of an active equivalent dose of idursulfase for four weeks failed to reduce the levels of CD68 and Trem2. Administration of ETV:IDS also reduced the accumulation of soluble Trem2 (sTrem2) in the CSF of treated IDS KO;TfR mu / hu KI mice ( Figure 11 ). These data indicate that ETV:IDS can attenuate microglial activity in addition to GAG and secondary lysosomal lipid accumulation in the brain.

[0936] Example 7. Construction of fusion proteins containing IDS.

[0937] Design and cloning

[0938] Design an IDS-Fc fusion protein, which contains (i) a fusion polypeptide that fuses the mature human IDS enzyme with a human IgG1 fragment including the Fc region ("IDS-Fc fusion polypeptide"), and (ii) a modified human IgG1 fragment that contains a mutation conferring transferrin receptor (TfR) binding in the Fc region ("modified Fc polypeptide"). Specifically, create an IDS-Fc fusion polypeptide in which the IDS fragment is fused to the N-terminus or C-terminus of the human IgG1 Fc region. In some cases, a linker is placed between the IDS and the IgG1 fragment to relieve any steric hindrance between the two fragments. In all constructs, the signal peptide from κ-chain V-III, amino acids 1-20 (UniProtKB ID–P01661) is inserted upstream of the fusion to facilitate secretion, and the IDS is truncated, consisting of amino acids S26-P550 (UniProtKB ID–P22304). The fragment of the human IgG1 Fc region used corresponds to amino acids D104-K330 of the sequence in UniProtKB ID P01857 (positions 221-447, EU numbering, including 10 amino acids of the hinge (positions 221-230)). In some embodiments, a second Fc polypeptide derived from human IgG1 residues D104-K330 but lacking the IDS fusion is co-transfected with the IDS-Fc fusion polypeptide to produce a heterodimeric fusion protein with one IDS enzyme ("single enzyme"). In some constructs, the IgG1 fragment contains additional mutations to promote heterodimerization of the two Fc regions. Similarly designed and constructed are control IDS-Fc fusion proteins lacking the mutations conferring TfR binding, except that these proteins lack the mutations conferring TfR binding. As an additional control, IDS (amino acids S26-P550) with a C-terminal hexahistidine tag (SEQ ID NO:203) was generated to facilitate detection and purification.

[0939] The IDS-Fc fusion protein that binds to TfR used in the examples is a dimer formed by an IDS-Fc fusion polypeptide and a modified Fc polypeptide that binds to TfR. For the dimer in which the IDS enzyme is linked to the N-terminus of the Fc region, the IDS-Fc fusion polypeptide may have the sequence of any one of SEQ ID NOs: 113, 193, and 197. In these sequences, the IDS sequence is underlined and contains a cysteine (double underlined) modified to formylglycine at position 59. The IDS is joined to the Fc polypeptide through a GGGGS linker (SEQ ID NO: 201). A part of the IgG1 hinge region (DKTHTCPPCP; SEQ ID NO: 111) is included at the N-terminus of the Fc polypeptide. The CH2 domain sequence begins at position 541 of SEQ ID NOs: 113, 193, and 197.

[0940] The IDS-Fc fusion protein ETV:IDS 35.21 is a dimer formed by an IDS-Fc fusion polypeptide having the sequence of any one of SEQ ID NOs: 113, 193, and 197 and a modified Fc polypeptide that binds to TfR and has the sequence of SEQ ID NO: 114. The first 10 amino acids are a part of the IgG1 hinge region. The CH2 domain sequence begins at position 11 of SEQ ID NO: 114.

[0941] The IDS-Fc fusion protein ETV:IDS 35.21.17.2 is a dimer formed by an IDS-Fc fusion polypeptide having the sequence of any one of SEQ ID NOs: 113, 193, and 197 and a modified Fc polypeptide that binds to TfR and has the sequence of SEQ ID NO: 190. The first 10 amino acids are a part of the IgG1 hinge region. The CH2 domain sequence begins at position 11 of SEQ ID NO: 190.

[0942] The IDS-Fc fusion protein ETV:IDS 35.23.2 is a dimer formed by an IDS-Fc fusion polypeptide having the sequence of any one of SEQ ID NOs: 113, 193, and 197 and a modified Fc polypeptide that binds to TfR and has the sequence of SEQ ID NO: 191. The first 10 amino acids are a part of the IgG1 hinge region. The CH2 domain sequence begins at position 11 of SEQ ID NO: 191.

[0943] The IDS-Fc fusion protein ETV:IDS 35.21.17 is a dimer formed by an IDS-Fc fusion polypeptide having a sequence of any one of SEQ ID NOs: 113, 193, and 197 and a modified Fc polypeptide that binds to TfR having a sequence of SEQ ID NO: 117. The N-terminus of the modified Fc polypeptide may include a portion of the IgG1 hinge region (e.g., SEQ ID NO: 111).

[0944] Recombinant Protein Expression and Purification

[0945] To express the recombinant IDS enzyme fused to the Fc region, ExpiCHO cells (Thermo Fisher Scientific) were transfected with the relevant DNA construct using the Expifectamine TM CHO transfection kit according to the manufacturer's instructions (Thermo Fisher Scientific). The cells were grown in ExpiCHO TM expression medium at 37 °C, 6% CO 2 and 120 rpm on an orbital shaker (Infors HT Multitron). Briefly, at 0.8 μg DNA plasmid per milliliter of culture volume, log-phase growing ExpiCHO 6 cells were transfected at a density of 6 × 10 TM cells per milliliter. After transfection, the cells were returned to 37 °C and the transfected cultures were fed as indicated within 18 - 22 hours after transfection. The transfected cell culture supernatant was harvested 120 hours after transfection by centrifugation at 3,500 rpm for 20 minutes. The clarified supernatant was filtered (0.22 μM membrane) and stored at 4 °C. Expression of the epitope-tagged IDS enzyme (used as a control) was performed as described above with minor modifications. Briefly, the IDS enzyme with a C-terminal hexahistidine tag (SEQ ID NO: 203) was expressed in ExpiCHO cells.

[0946] Purify the IDS-Fc fusion protein with (or without) an engineered Fc region conferring TfR binding from cell culture supernatants using protein A affinity chromatography. Load the supernatant onto a HiTrap MabSelect SuRe protein A affinity column (GE Healthcare Life Sciences, using an Akta Pure system). Then wash the column with >20 column volumes (CV) of PBS. Elute the bound protein using 100 mM citrate / NaOH buffer pH 3.0 (containing 150 mM NaCl). Immediately after elution, neutralize the fractions using 1 M arginine-670 mM succinate buffer pH 5.0 (diluted 1:5). Assess the homogeneity of the IDS-Fc fusion protein in the eluted fractions by reducing and non-reducing SDS-PAGE.

[0947] To purify the IDS enzyme with a hexahistidine tag (SEQ ID NO:203), dialyze the transfected supernatant against 15 L of 20 mM HEPES pH 7.4 containing 100 mM NaCl overnight. Bind the dialyzed supernatant to a HisTrap column (GE Healthcare Life Sciences, using an Akta Pure system). After binding, wash the column with 20 CV of PBS. Elute the bound protein using PBS containing 500 mM imidazole. Assess the homogeneity of the IDS enzyme in the eluted fractions by reducing and non-reducing SDS-PAGE. Dilute the pooled fractions containing the IDS enzyme 1:10 in 50 mM Tris pH 7.5 and further purify using QSepharose High Performance (GE Healthcare). After binding, wash the column with 10 CV of 50 mM Tris pH 7.5. Elute the bound protein using a linear gradient to 50 mM Tris pH 7.5 and 0.5 M NaCl and collect in 1 CV fractions. Assess fraction purity by non-reducing SDS-PAGE. Purification yields homogeneous IDS-Fc fusion protein and IDS enzyme with a hexahistidine tag (SEQ ID NO:203).

[0948] Example 8. Effect of peripherally administered ETV:IDS on GAG and neurofilament light chain (Nf-L) in IDS KO×TfR muhu mice

[0949] Examine the effect of weekly intravenous administration of ETV:IDS on GAG and neurofilament light chain (Nf-L) in IDS KO×TfR muhu mice.

[0950] Materials and methods

[0951] Animal care

[0952] Mice were housed on a 12-hour light / dark cycle and had free access to water and a standard rodent diet ( #25502, irradiated).

[0953] Mouse strains

[0954] The IDS KO×TfR mu / hu mice and TfR mu / hu KI mice used in this study were described in Example 1 above. All mice used in this study were male.

[0955] Administration and sample collection

[0956] Eight-week-old IDS KO; TfR mu / hu KI mice were intravenously (IV) administered 1 mg / kg or 3 mg / kg ETV:IDS via the tail vein. Eight (8)-week-old TfR mu / hu KI mice injected with vehicle (IDS WT) were used as non-disease controls. ETV:IDS or vehicle was administered once a week for 13 weeks. All animals were euthanized 7 days after the last administration.

[0957] Serum and terminal CSF samples were collected as described in Example 1 during life. Brain and liver tissue samples were also collected as described in Example 1. In addition, urine was collected immediately before termination and cooled. Then urine samples were stored in a refrigerator set to maintain at -60°C to -80°C for urine biomarker analysis.

[0958] Quantification of GAG

[0959] Brain and liver tissues were prepared to quantify GAG (e.g., heparan sulfate (HS) and dermatan sulfate (DS)) as described in Example 1. Before LCMS assay to quantify GAG, protein lysates (from tissues) or CSF, urine, or serum were mixed with a combination of heparinases I, II, III, and chondroitinase B. The digest was mixed with acetonitrile and analyzed by LCMS. LCMS assay was performed as described in Example 1 to quantify GAG.

[0960] Methods for CSF and serum analysis of Nf-L

[0961] Nf-L levels in serum and CSF were measured as described in Example 2.

[0962] Results

[0963] GAG in brain, CSF, liver, and urine. Measured 7 days after the last dose of ETV:IDS from IDS KO; TfR mu / huGAG levels in the brains of mice and compared with vehicle-treated and TfR mu / hu mice. Consistent with earlier results, brain GAG values decreased with increasing ETV:IDS dose and, relative to vehicle-treated IDS KO; TfR mu / hu mice, the treatment efficiencies at 1 mg / kg and 3 mg / kg were 64% and 75%, respectively (data not shown). CSF GAG values also decreased with increasing ETV:IDS dose and, relative to vehicle-treated IDS KO; TfR mu / hu mice, the treatment efficiencies at 1 mg / kg and 3 mg / kg were 60% and 70%, respectively (data not shown). Liver GAG levels were nearly completely corrected at all dose levels of ETV:IDS (relative to vehicle-treated IDS KO; TfR mu / hu mice, the treatment efficiencies at 1 mg / kg and 3 mg / kg were 98% and 96%, respectively) ( Figure 28 ). After 13 weeks of weekly treatment with ETV:IDS, GAG levels in urine also decreased. The treatment efficiencies of creatinine-normalized GAG levels at 1 mg / kg and 3 mg / kg were 78% and 85%, respectively ( Figure 28 ).

[0964] CSF neurofilament light chain (Nf-L). As Figures 26A - 26C shown, elevated Nf-L levels in CSF and serum were observed in a mouse model of Hunter syndrome. Weekly doses of ETV:IDS were able to reverse these changes over a 13-week period, and complete normalization of CSF Nf-L was observed in IDS KO; TfR mu / hu treated with ETV:IDS. The treatment efficiencies of Nf-L correction at 1 mg / kg and 3 mg / kg were 82% and 117%, respectively, reducing the level of Nf-L to that observed in IDS WT controls ( Figure 27 ).

[0965] Table

[0966] Table 1. Summary of GlcCer levels in the brains of IDS KO mice treated with ETV:IDS (fold relative to WT)

[0967] KO + vehicle KO + ETV:IDS KO + idursulfase GlcCer(d18:1, 16:0) 2.2 1.2 1.8 GlcCer(d18:1, 18:0) 1.5 1.0 1.4 GlcCer(d18:2, 18:0) 1.6 0.9 1.3 GlcCer(d18:1, 20:0) 1.6 1.1 1.4 GlcCer(d18:2, 20:0) 1.7 1.1 1.4 GlcCer(d18:1, 22:0) 2.0 1.1 1.8 GlcCer(d18:1, 22:1) 1.2 1.1 1.1 GlcCer(d18:2, 22:0) 1.1 1.0 1.3 GlcCer(d18:1, 24:1) 1.6 1.1 1.4 GlcCer(d18:1, 24:0) 1.9 1.1 1.6

[0968] Table 2. Lipid levels in the brains of IDS KO mice treated with vehicle, ETV:IDS, or Elaprase (fold relative to WT)

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[1014] All publications, patents, and patent documents are hereby incorporated by reference in their entirety, as if each were incorporated by reference individually. The present disclosure has been described with reference to various specific and preferred embodiments and techniques. However, it is to be understood that many variations and modifications can be made while remaining within the spirit and scope of the invention.

Claims

1. A method for detecting one or more biomarkers in a subject with lysosomal storage disease (LSD), the method comprising: 1) Measuring the concentration of a combination of two or more lipids in a sample from the subject, wherein the combination of lipids is selected from the group consisting of: a) Bis(monoacylglycerol) phosphate (BMP); b) GM2 ganglioside and / or GM3 ganglioside; c) GD3 ganglioside; d) GD1a / b ganglioside; and e) Glucosylceramide (GlcCer); 2) Measuring the concentration of GlcCer in a sample from the subject, provided that the LSD is a mucopolysaccharidosis (MPS) disorder; 3) Measuring the concentration of neurofilament light chain (Nf-L) in a sample from the subject; and / or 4) Measuring the concentration of soluble triggering receptor expressed on myeloid cells 2 (sTREM2) in a sample from the subject.

2. A method for evaluating the therapeutic efficacy in a subject with LSD, the method comprising: 1) Measuring the concentration of a combination of two or more lipids in a sample obtained from the subject after administration of the treatment, wherein the combination of lipids is selected from the group consisting of: a) BMP; b) GM2 ganglioside and / or GM3 ganglioside; c) GD3; d) GD1a / b; and e) GlcCer; 2) Measuring the concentration of GlcCer in a sample obtained from the subject after administration of the treatment, provided that the LSD is an MPS disorder; 3) Measuring the concentration of Nf-L in a sample obtained from the subject after administration of the treatment; and / or 4) Measuring the concentration of sTREM2 in a sample obtained from the subject after administration of the treatment; wherein a decrease in the concentration of the selected lipid / protein in the sample obtained from the subject after administration of the treatment, compared to the concentration of the selected lipid / protein in the sample obtained from the subject before administration of the treatment, is correlated with therapeutic efficacy.

3. The method according to claim 1 or 2, the method further comprising administering an LSD treatment to the subject.

4. The method according to any one of claims 1-3, the method further comprising adjusting the treatment regimen of the subject.

5. A method for treating LSD in a subject, the method comprising: 1) Administering an LSD treatment to the subject; 2) Measuring the concentration of: a) A combination of two or more lipids in a sample from the subject, wherein the combination of lipids is selected from the group consisting of: i) BMP; ii) GM2 ganglioside and / or GM3 ganglioside; iii) GD3; iv) GD1a / b; and v) GlcCer; b) GlcCer in a sample from the subject, provided that the LSD is an MPS disorder; c) Nf-L in a sample from the subject; and / or d) sTREM2 in a sample from the subject; and 3) Adjust the dose of the LSD treatment based on the concentration of the selected lipid / protein in the sample from the subject as compared to a control value.

6. The method according to any one of claims 1-5, the method comprising measuring the concentration of a combination of two or more lipids.

7. The method according to any one of claims 1-5, the method comprising measuring the concentration of sTREM2.

8. The method according to any one of claims 1-5, the method comprising measuring the concentration of Nf-L.

9. The method according to any one of claims 1-5, the method comprising measuring the concentration of GlcCer, wherein the LSD is an MPS disorder.

10. The method according to any one of claims 1-5, the method comprising measuring the concentration of one or more lipids and the concentration of sTREM2.

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