EP4669361A2

EP4669361A2 - BMP synthase actuators and inhibitors

Info

Publication number: EP4669361A2
Application number: EP24761084.3A
Authority: EP
Inventors: Monther ABU-REMAILEH; Uche MEDOH; Jian Xiong
Original assignee: Leland Stanford Junior University
Current assignee: Leland Stanford Junior University
Priority date: 2023-02-24
Filing date: 2024-02-23
Publication date: 2025-12-31
Also published as: WO2024178352A3; WO2024178352A2

Abstract

Disclosed herein are methods, compositions, and agents directed to the modulation of the level and/or the activity of bis(monoacylglycero)phosphate (BMP) synthase, particularly for maintaining lysosomal function, increasing lipid catabolism, increasing cholesterol trafficking, and treating diseases and disorders in a subject (e.g., neurodegenerative diseases, proliferative diseases, and the like).

Description

BMP SYNTHASE ACTIVATORS AND INHIBITORS

TECHNICAL FIELD

[0001] Disclosed herein are methods, compositions, and agents directed to the modulation of the level and/or the activity of bis(monoacylglycero /phosphate (BMP) synthase, particularly for maintaining lysosomal function, increasing lipid catabolism, increasing cholesterol trafficking, and treating diseases and disorders in a subject (e.g., neurodegenerative diseases, proliferative diseases, and the like).

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63/448,128, filed February 24, 2023, the content of which is herein incorporated by reference in its entirety.

SEQUENCE LISTING STATEMENT

[0003] The content of the electronic sequence listing titled STDU2-41553-601_SQL (Size:

16,149 bytes; and Date of Creation: February 23, 2024) is herein incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0004] This invention was made with Government support under AG066515 awarded by the National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND

[0005] Bis(monoacylglycero)phosphate (BMP), an anionic, late endosome/lysosome (LE/LY)-specific glycerophospholipid with unusual esterification, unsaturation, and stereochemistry, has been shown to potently activate lipid catabolism on intraluminal vesicles (ILVs), promote lysosomal cholesterol egress, and regulate the formation of ILVs in LE/LYs to maintain cellular homeostasis. Notably, lysosomal dysfunction is a hallmark of neurodegeneration, and aberrant abundances of BMP have been reported in rare and common neurodegenerative diseases, such as lysosomal storage diseases (LSDs), Alzheimer’s disease, Parkinson’s disease, and frontotemporal dementia (FTD). Beyond neurodegeneration, studies additionally implicate BMP-dependent pathways in the development of atherosclerosis, drug- induced phospholipidosis, viral infection, endosomal escape, and cancer. Given the stimulatory effect of BMP on lysosomal function, studies suggest BMP accumulation in certain diseases may be an ameliorative response to lysosomal dysfunction. However, BMP synthesis and regulation is poorly understood, thereby confounding studies and advances in understanding the etiology and potential treatment methods for these diseases.

SUMMARY

[0006] In one aspect, disclosed herein are methods of stimulating and/or maintaining lysosomal function in a subject in need thereof comprising increasing the level and/or the activity bis(monoacylglycero)phosphate (BMP) synthase in the subject. In some embodiments, the methods comprise administering to the subject an effective amount of a BMP synthase activator or a composition thereof and/or a BMP synthase, a catalytically active fragment or variant thereof, or a nucleic acid encoding BMP synthase or a catalytically active fragment or variant thereof. In some embodiments, the methods comprise administering to the subject an effective amount of a BMP synthase activator or a composition thereof. In some embodiments, the methods comprise administering to the subject an effective amount of a BMP synthase, a catalytically active fragment or variant thereof, or a nucleic acid encoding BMP synthase or a catalytically active fragment or variant thereof. In some embodiments, the activator comprises a small molecule activator of BMP synthase. In some embodiments, the activator comprises progranulin, granulin peptides, or derivatives thereof. In some embodiments, the activator comprises phosphatidyl glycerol or derivatives thereof, lysophosphatidylglycerol or derivatives thereof, or combinations thereof. In some embodiments, the activator comprises a protease inhibitor (e.g., a cysteine protease inhibitor).

[0007] In some embodiments, the BMP synthase comprises an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%) identity to SEQ ID NO: I. In some embodiments, the BMP synthase comprises an amino acid sequence of SEQ ID NO: 1. In some embodiments, the BMP synthase comprises an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%) identity to SEQ ID NO: 2. In some embodiments, the BMP synthase comprises an amino acid sequence of SEQ ID NO: 2.

[0008] In some embodiments, the BMP synthase activator or BMP synthase or catalytically active fragment thereof is linked to a polypeptide configured to bind to a blood-brain barrier (BBB) receptor. In some embodiments, the polypeptide comprises a receptor-binding domain from an apolipoprotein.

[0009] In some embodiments, the BMP synthase activator or BMP synthase or catalytically active fragment thereof is linked to a polypeptide comprising a first Fc region. In some embodiments, the first Fc region is derived from an immunoglobulin IgG Fc region. In some embodiments, the polypeptide is an antibody or fragment thereof. In some embodiments, the antibody or fragment thereof is configured to bind to a blood-brain barrier (BBB) receptor. [0010] In some embodiments, the methods further comprise administering a polypeptide comprising a second Fc region configured to form a dimer with the first Fc region.

[0011] In some embodiments, the methods further comprise activating autophagy or autophagy related pathways, increasing the level and/or the activity of transcription factor EB (TFEB), or a combination thereof.

[0012] In some embodiments, the methods further comprise administering at least one immune modulator or neuroprotective compound.

[0013] In some embodiments, the subject has or is suspected of having a disease or disorder characterized by lysosomal dysfunction, decreased lipid catabolism, and/or increased cholesterol accumulation.

[0014] In some embodiments, the subject has or is suspected of having a neurodegenerative disease. In some embodiments, the neurodegenerative disease is selected from Niemann-Pick disease, frontotemporal dementia (FTD), Parkinson’s disease, Alzheimer’s disease, a neuronal ceroid lipofuscinosis, amyotrophic lateral sclerosis (ALS), and Dementia with Lewy Bodies (DLB). In some embodiments, the neuronal ceroid lipofuscinosis is selected from neuronal ceroid lipofuscinosis type 1 (CLN1), neuronal ceroid lipofuscinosis type 2 (CLN2), neuronal ceroid lipofuscinosis type 3 (CLN3), neuronal ceroid lipofuscinosis type 4 (CLN4), neuronal ceroid lipofuscinosis type 5 (CLN5), neuronal ceroid lipofuscinosis type 6 (CLN6), neuronal ceroid lipofuscinosis type 7 (CLN7), neuronal ceroid lipofuscinosis type 8 (CLN8), neuronal ceroid lipofuscinosis type 9 (CLN9), neuronal ceroid lipofuscinosis type 10 (CLN10), neuronal ceroid lipofuscinosis type 11 (CLN11), neuronal ceroid lipofuscinosis type 12 (CLN12), neuronal ceroid lipofuscinosis type 13 (CLN13), and neuronal ceroid lipofuscinosis type 14 (CLN14). [0015] In some embodiments, the subject has or is suspected of having a lysosomal storage disorders. In some embodiments, the lysosomal storage disorder is selected from: Gaucher’s disease, metachromatic leukodystrophy, GM1 gangliosidosis, Tay-Sachs disease, Sandhoff disease, Fabry disease, Farber disease, Wolman disease, mucolipidosis Types I, II/III and IV, Type I mucopolysaccharidoses, Type II mucopolysaccharidoses, Type III mucopolysaccharidoses, Type IV mucopolysaccharidoses, Type VI mucopolysaccharidoses, and Krabbe disease.

[0016] In some embodiments, the subject has or is suspected of having drug-induced phospholipidosis, familial hypercholesterolemia (FH), a cardiovascular disease, atherosclerosis, obesity, a fatty liver disease, or any combination thereof.

[0017] In some embodiments, the administration is via the cerebrospinal fluid. In some embodiments, the administration is intrathecal, intracistemal, or intracerebroventricular. In some embodiments, the administration comprises systemic administration.

[0018] In another aspect, disclosed herein are methods for treating or preventing a disease or disorder in a subject comprising administering to the subject a therapeutically or prophylactically effective amount of a BMP synthase inhibitor or a composition thereof to the subject. In some embodiments, the disease or disorder is selected from a Von Hippel-Lindau (VHL) related cancer, K-Ras driven cancers, lung cancer, pancreatic cancer, prostate cancer, breast cancer, cancers related to low levels of HSP70, or an infectious disease.

[0019] In some embodiments, the inhibitor is selected from the group consisting of a protein configured to bind BMP synthase or a substrate thereof, a gene silencing oligonucleotide, a small molecule inhibitor of BMP synthase, a negative allosteric modulating agent, a protease, and combinations thereof.

[0020] In some embodiments, the inhibitor is a dominant negative BMPS or variant or fragment thereof. In some embodiments, the dominant negative BMPS or variant or fragment thereof comprises one or more mutations in the catalytic active site. In some embodiments, the dominant negative BMPS or variant or fragment thereof comprises one or more mutations in cysteine 231, histidine 117, & glutamate 134 in reference to SEQ ID NO: 1.

[0021] In some embodiments, the BMP synthase inhibitor comprises a negative allosteric modulating agent. In some embodiments, the negative allosteric modulating agent is configured to bind to a binding site of BMP synthase, a catalytically active fragment or variant thereof, comprising residues at positions 73, 143, and 296 in reference to SEQ ID NO: 1 . In some embodiments, the binding site comprises K73, N143, and K296, in reference to SEQ ID NO: 1. [0022] In some embodiments, the BMP synthase inhibitor comprises a glycerophosphodiester. In some embodiments, the inhibitor is selected from the group consisting of glycerophosphatidylserine (GPS), glycerophosphatidylethanolamine (GPE), glycerophosphoglycerol (GPG), glycosylphosphatidylinositol (GPI), glycerophosphatidylcholine (GPC), and combinations thereof.

[0023] In yet another aspect, disclosed herein are methods for treating or preventing a disease or disorder in a subject, comprising administering to the subject an effective amount of BMP synthase, a catalytically active fragment or variant thereof, or a nucleic acid encoding BMP synthase, a catalytically active fragment or variant thereof to the subject.

[0024] In some embodiments, the disease or disorder is characterized by lysosomal dysfunction, decreased lipid catabolism, and/or increased cholesterol accumulation.

[0025] In some embodiments, the disease or disorder is a neurodegenerative disease. In some embodiments, the neurodegenerative disease is selected from Niemann-Pick disease, frontotemporal dementia (FTD), Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), and Dementia with Lewy Bodies (DLB). In some embodiments, the neurodegenerative disease is selected from neuronal ceroid lipofuscinosis type 1 (CLN1), neuronal ceroid lipofuscinosis type 2 (CLN2), neuronal ceroid lipofuscinosis type 3 (CLN3), neuronal ceroid lipofuscinosis type 4 (CLN4), neuronal ceroid lipofuscinosis type 6 (CLN6), neuronal ceroid lipofuscinosis type 7 (CLN7), neuronal ceroid lipofuscinosis type 8 (CLN8), neuronal ceroid lipofuscinosis type 9 (CLN9), neuronal ceroid lipofuscinosis type 10 (CLN10), neuronal ceroid lipofuscinosis type 11 (CLN11), neuronal ceroid lipofuscinosis type 12 (CLN12), neuronal ceroid lipofuscinosis type 13 (CLN13), and neuronal ceroid lipofuscinosis type 14 (CLN14).

[0026] In some embodiments, the subject has or is suspected of having a lysosomal storage disorders. In some embodiments, the lysosomal storage disorder is selected from: Gaucher’s disease, metachromatic leukodystrophy, GM1 gangliosidosis, Tay-Sachs disease, Sandhoff disease, Fabry disease, Farber disease, Wolman disease, mucolipidosis Types I, II/III and IV, Type I mucopolysaccharidoses, Type II mucopolysaccharidoses, Type III mucopolysaccharidoses, Type IV mucopolysaccharidoses, Type VI mucopolysaccharidoses, and Krabbe disease.

[0027] In some embodiments, the subject has or is suspected of having drag-induced phospholipidosis, familial hypercholesterolemia (FH), a cardiovascular disease, atherosclerosis, obesity, a fatty liver disease, or any combination thereof.

[0028] In some embodiments, the BMP synthase comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 1. In some embodiments, the BMP synthase or catalytically active fragment thereof is linked to a polypeptide configured to bind to a blood-brain barrier (BBB) receptor. In some embodiments, the polypeptide comprises a receptor-binding domain from an apolipoprotein.

[0029] In some embodiments, the BMP synthase or catalytically active fragment thereof is linked to a polypeptide comprising a first Fc region. In some embodiments, the first Fc region is derived from an immunoglobulin IgG Fc region. In some embodiments, the polypeptide is an antibody or fragment thereof. In some embodiments, the antibody or fragment thereof is configured to bind to a blood-brain barrier (BBB) receptor.

[0030] In some embodiments, the methods further comprise administering a polypeptide comprising a second Fc region configured to form a dimer with the first Fc region.

[0031] In some embodiments, the administration is via the cerebrospinal fluid. In some embodiments, the administration is intrathecal, intracistemal, or intracerebroventricular. In some embodiments, the administration comprises systemic administration.

[0032] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description and accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIGS. 1A-1D show CLN5 deficiency results in BMP depletion and lysophosphatidylglycerol (EPG) accumulation. FIG. 1A is untargeted lipid analysis of HEK293T lysosomes upon CLN5 loss. Data presented as a volcano plot of log2-transformed fold change in the abundance of lipids between CLN5 knockout and wildtype HEK293T lysosomes.

Significantly altered lipids include EPG (red) and bis(monoacylglycero)phosphate (BMP, yellow). BMP/phosphatidylglycerol (PG) (yellow) annotation indicates compounds for which fragmentation data was not acquired. Horizontal line indicates a p-value of 0.05, and vertical line indicates a fold change of 2. Each genotype measurement represents n = 4 biologically independent samples. P-values were calculated by ANOVA with Tukey HSD test and corrected by the Bcnjamini-Hochbcrg method with an FDR = 5%. FIGS. 1B-1C arc targeted analyses of BMP and LPG in whole cells and lysosomes. Fold changes in lysosomal BMPs and LPGs between CLN5 knockout and wildtype HEK293T cells were calculated after subtracting background from control samples and normalizing to endogenous lipid. If species is not detected in wildtype cells, normalized abundance is presented. ND, not detected. WT, wildtype. Data presented as mean ± SD of n = 4 biologically independent samples. FIG. ID is targeted lipid analysis of LPG and BMP normalized abundances from CLN5 -deficient iPSCs and iNeurons compared with the WT. Plotted values are normalized to endogenous lipids. Data presented as mean of n = 3 biologically independent samples. ND, not detected. WT, wild-type.

[0034] FIGS. 2A-2K show CLN5 is bis(monoacylglycero)phosphate synthase (BMPS). FIGS. 2A and 2B show that recombinant CLN5 synthesizes BMP with representative extracted ion chromatograms (EIC) for LPG (18:1) and BMP (18:1/18:1) from a reaction between recombinant CLN5 and LPG (18:1) under acidic conditions. FIG. 2C shows BMPS activity results in glycerophosphoglycerol (GPG) release with representative EIC for GPG byproduct release. FIG. 2D is MS/MS spectral confirmation of BMP (18:1/18:1) synthesis compared to commercial standard (18: 1/18:1) BMP (S,S) as drawn. FIG. 2E, left is a representative Michaelis-Menten curve (MM) for BMPS activity towards LPG (18:1) using GPG to monitor the reaction. FIG. 2E, right is a Lineweaver-Burk transformation of MM curve. The experiment was performed at least three times. K_cat (catalytic rate constant), K_m (Michaelis constant), & V_max (maximum velocity). FIG. 2F is a schematic for recombinant BMPS (rBMPS) supplementation experiment. HEK293T conditioned media were supplemented with rBMPS for 48 hours prior to lipid analysis. FIG. 2G shows rBMPS (rCLN5) rescues BMP deficiency and LPG storage. Fold changes in levels of BMP and LPG species normalized to endogenous lipid. Data presented as mean ± SD of n = 4 biologically independent samples. FIG. 2H shows BMPS interacts with BMP liposomes. Recombinant BMPS was incubated with 75:25 %mol l-palmitoyl-2- oleoylphosphatidylcholine (POPC):BMP (18:1/18:1) liposomes under acidic and neutral conditions until reaction reached equilibrium tested by repeated temporal measurements. Data presented as mean ± SE of n = 3 technical replicates. The experiment was performed at least three times. FIG. 21 shows BMP-laden liposomes stimulate BMP synthesis. Recombinant BMPS was incubated with either 100 %mol POPC or 75:25 %mol POPC/BMP (18:1/18:1) liposomes containing equimolar LPG (18: 1). Representative graph shown for experiment performed at least three times. FIG. 2J shows amiodarone inhibits BMPS activity towards monomeric and liposomal LPG. Recombinant BMPS was incubated with either monomeric LPG (18:1) or liposomal LPG (18:1) in the presence or absence of amiodarone. Data presented as mean ± SD of n = 4 biologically independent samples. FIGS. 2K is a diagram for BMPS activity at a lipid:water interface. BMP-laden vesicles enhance BMPS activity towards LPG. This activation is significantly inhibited by cationic amphiphilic drugs (CADs) like amiodarone.

[0035] FIGS. 3A-3E show that an active site thiol mediates base exchange for BMP synthesis. FIG. 3A shows docking of one stereoisomer of LPG (18:1) onto an experimental CLN5 structure (PDB 6R99). Predicted catalytic triad residues C231, Hl 17, and E134 are annotated. FIG. 3B shows that weaker serine nucleophile attenuates BMPS activity, as measured by monitoring GPG release. Recombinant BMPS (rCLN5) WT and C231S were incubated with LPG (18:1) under acidic conditions. Data presented as mean ± SD of n = 3 biologically independent samples. FIGS. 3C and 3D show that rCLN5 C231S (rCLN5 CS) exhibits reduced rescue of LPG storage and BMP deficiency. Fold change in levels of BMP and LPG species normalized to endogenous lipid. Data presented as mean ± SD of n - 4 biologically independent samples. FIG. 3E is a diagram of the BMPS (CLN5) reaction mechanism. The BMPS active site thiol substitutes at the LPG carbonyl carbon releasing a GPG byproduct and abstracting an acyl chain to form a high-energy enzyme-acyl intermediate. Through a base-exchange reaction, the electrophilic intermediate is substituted at its carbonyl carbon by an LPG alcohol to complete the catalytic cycle and release BMP.

[0036] FIGS. 4A-4L show late endosomes/lysosomes (LE/LY) synthesize BMP to maintain lipid homeostasis. FIG. 4A is a schematic for LE/LY delivery of deuterated tracers for metabolism and LC-MS/MS analysis. Deuterated lipids are conjugated to BSA and supplemented in HEK293T conditioned media after starvation to increase endocytic flux. After several time points, lipids were extracted for LC-MS/MS analysis. FIG. 4B is a depiction of monitored deuterated phosphatidylglycerol (PG) metabolites bearing 5 deuterium atoms in their glycerol moiety. FIG. 4C shows that CLN5 knockout HEK293Ts are unable to synthesize deuterated BMP. Intensities for d5-BMP (18:1/18:1) in CLN5 knockout and wildtype HEK293Ts are presented after background subtraction and normalization to endogenous lipids. Data presented as mean ± SD of n = 3 biologically independent samples. FIG. 4D is a schematic for an exemplary recombinant BMPS (rCLN5) complementation experiment in BMPS deficient lysosomal protein extract. Lysosomes were immunoprecipitated and harvested for proteins. Lysosomal proteins were supplemented with rCLN5 and supplied with d5-PG (16:0/18:1) under acidic conditions. FIG. 4E shows that lysosomes require rCLN5 to synthesize BMP. Fold change in the levels of deuterated BMP species were monitored. Data presented as mean ± SD of n - 3 biologically independent samples. FIG. 4F is a depiction of monitored deuterated phosphatidylcholine (PC) metabolites bearing 9 deuterium atoms in their methyl groups. FIG. 4G shows CLN5 knockout HEK293T lysosomes exhibit impaired phospholipid catabolism. Intensities for d9-LPC (18:0) production in CLN5 knockout and wildtype HEK293Ts are presented after background subtraction and normalization to endogenous lipids. Data presented as mean ± SD of n = 3 biologically independent samples. FIG. 4H shows CLN5 deficient HEK293T cells accumulate cholesterol and the effect of rCLN5 supplementation. Filipin staining of cholesterol in wildtype, CLN5 knockout HEK293Ts, and rCLN5 supplemented CLN5 knockout HEK293Ts were determined and are presented relative to wildtype. Representative data shown for experiment repeated at least three times with 20 cells per condition. FIG. 41 is a depiction of monitored deuterated glucosylceramide metabolites bearing 5 deuterium atoms in their hydrocarbon chain. FIG. 4J shows that CLN5 knockout HEK293T lysosomes exhibit impaired GCase (Glucocerebrosidase) activity. Intensities for d5-HexlCer (dl8: 1/18:0) production in CLN5 knockout and wild-type HEK293T cells are presented after background subtraction and normalization to endogenous lipids. Experiment was done as in FIG. 4A. Data presented as mean ± SD of n = 3 biologically independent samples. FIG. 4K shows CLN5- deficient iNeurons accumulate GM3 gangliosides. Fold changes in lipid species abundance between CLN5 knockout and wild-type HEK293T cells were calculated after subtracting background from control samples and normalizing to endogenous lipids. Data presented as mean ± SD of n = 3 biologically independent samples. FIG. 4L shows BMPS-deficient HEK293Ts have fewer ILVs. Representative electron micrographs of CLN5 knockout and wild-type HEK293T cells. The average number of ILVs within each identified endolysosome per cell are counted with 10 cells per condition. Arrows indicate ILVs. Scale bars, 2 pm. Inset scale bar, 1 pm. EL, endo-lysosome; M, mitochondria.

[0037] FIGS. 5 A and 5B show characterization and validation of CLN5 knockout cells. FIG. 5A is western blot analysis of CLN5 knockout and wildtype HEK293Ts. CLN5 knockout cells are deficient in the CLN5 gene product. FIG. 5B is a graph of the fold changes of endogenous PC (18:1/18:1) lipid used for normalization in targeted lipid analysis. Data presented as mean ± SD of n = 4 biologically independent samples. FIG. 5C is targeted analysis of PG EIC intensities derived from untargeted experiment in FIG. 1A. Fold changes in lipid species between CLN5 knockout and wildtype HEK293T lysosomes were calculated after subtracting background from control samples and normalizing to endogenous lipid. Data presented as mean ± SD of n = 4 biologically independent samples. FIG. 5D is Western blot analysis of CLN5 knockout and wildtype iNeurons.

[0038] FIGS. 6A-6I show the purification of recombinant BMPS protein. FIGS. 6A and 6B show the purification of His-tagged, recombinant WT BMPS protein (CLN5). FIG. 6A is a Coomassie- stained SDS-PAGE gel. FIG. 6B is size exclusion chromatography purification of a multimeric protein. FIGS. 6C-6E show the purification of CLN5 mutants with is a Coomassie- stained SDS-PAGE gel. FIG. 6F shows bands (duplicate) identified in Coomassie stain corresponding to recombinant BMPS protein. FIG. 6G shows staining of recombinant BMPS protein is concentration dependent. FIG. 6H shows that recombinant BMPS protein possesses weak thioesterase activity. 1 pg BMPS protein was incubated with 10 pM MU-6S-palm-pGlc in 50 mM Sodium Acetate: Acetic Acid pH 5.0, 150 mM NaCl, and 20 pg 0-glucosidase for 30 minutes at 37 °C. Exact initial velocities were determined from a 4-methylumbelliferyl standard curve. Data presented as mean ± SD of n = 3 biologically independent samples. FIG. 61 shows purification of N143S BMPS protein.

[0039] FIGS. 7A-7E show additional biochemical characterization of BMPS. FIG. 7A is a representative Michaelis-Menten curve and Lineweaver-Burk plot for BMPS activity with monitoring of BMP formation. The experiment was performed at least three times. FIG. 7B is a representative graph for BMPS activity when recombinant BMPS incubated with LPG (18:1) at the indicated pH values. BMPS displays a slightly acidic pH optimum. FIG. 7C shows BMPS can utilize diverse LPG species. Recombinant BMPS (CLN5) was incubated with different LPG species and release of GPG was monitored. ND, not detected. Data presented as mean ± SD of n = 3 biologically independent samples. FIG. 7D is a graph showing recombinant BMPS 5 without a poly histidine tag (WTcieaved) retains BMP synthesis activity. LPG (18:1) was incubated with no enzyme or WTcieaved under acidic conditions. Data presented as mean ± SD of n = 3 biologically independent samples. FIG. 7E shows a representative Michaelis-Menten curve and Lineweaver- Burk plot for BMPS activity at pH 6.5 with GPG monitoring.

[0040] FIGS. 8A-8D show BMPS mutants exhibit comparable uptake during enzyme replacement. FIG. 8A is a schematic for fluorescently labeled, recombinant BMPS (CLN5) uptake experiments. Fluorescent wildtype or C231S mutant CLN5 were supplemented into CLN5 knockout HEK293T conditioned media for 48 hours and imaged. FIG. 8B shows fluorescence images of fluorescent CLN5 protein (Alexa488, green) and lysosomes (Lysotracker, red) after addition of fluorescent wildtype or C231S mutant BMPS to CLN5 knockout HEK293T cells. Scale bar = 10 pm. Representative image shown for experiment repeated at least three times. FIGS. 8C and 8D show C231S CLN5 exhibits no difference in cellular uptake and lysosomal localization compared to that of wildtype. Fluorescent CLN5 intensities and colocalization with Lysotracker in CLN5 knockout HEK293Ts were determined. Intensities are presented relative to wildtype. Twenty cells were analyzed per condition for intensity measurements. Ten images were analyzed per condition for colocalization analysis.

[0041] FIGS. 9A and 9B show recombinant BMPS (rCLN5) rescues BMP depletion and LPG storage after CLN5 knockout. Fold change in the levels of BMP and LPG species normalized to endogenous lipid. Data presented as mean ± SD of n = 4 biologically independent samples.

[0042] FIGS. 10A-10D show a cationic amphipathic helix mediates BMPS docking onto BMP-laden liposomes. FIG. 10A shows recombinant BMPS does not dock onto POPC liposomes. Recombinant BMPS was incubated with 100 %mol POPC liposomes at acidic pH followed by assessment of binding by microscale thermophoresis (MST). Data presented as mean ± SE of n = 3 technical replicates. The experiment was performed at least three times. NBD, no binding detected. FIG. 10B shows multiple sequence alignment of human CLN5 orthologs. Residues highlighted in blue and gray depict positive charge and hydrophobic character, respectively, at acidic pH. FIG. 10C is a depiction of BMPS cationic amphipathic helix (CAH). CAH is colored in red with basic and hydrophobic residues shown. FIG. 10D shows mutation of basic residues to acidic residues attenuates BMPS docking onto BMP liposomes. Recombinant BMPS mutants were incubated with 75:25 %mol POPC:BMP (18:1/18:1) liposomes under acidic conditions until reaction reached equilibrium tested by repeated temporal measurements of binding by microscale thermophoresis (MST). Data presented as mean ± SE of n = 3 technical replicates. The experiment was performed at least three times.

[0043] FIGS. 11A-11C are representative melting curves that show wildtype and mutant recombinant BMPS (CLN5) are thermally stable.

[0044] FIGS. 12A-12C are representative circular dichroism graphs showing that mutant recombinant BMPS (CEN5) retain similar secondary structure to that of wildtype.

[0045] FIGS. 13A-13C show substrate surface dilution limits BMPS activation by BMP- enriched liposomes. FIG. 13A is a depiction of lipid surface dilution. At a fixed lipid substrate concentration with full integration into liposomes, the enzyme activity decreases as the concentration of mixed liposomes increases. FIG. 13B shows BMP liposome stimulation of BMPS activity is limited by surface dilution. Recombinant BMPS was incubated with a fixed concentration of EPG (18:1) and increasing concentrations of 100 %mol POPC and 75:25 %mol POPC:BMP liposomes as to increase the liposome to EPG ratio. FIG. 13C shows anionic phospholipids activate BMPS activity. Recombinant BMPS was incubated with LPG (18:1/18:1) alone or liposomes containing PC (16:0/18:1), LPG (18:1/18:1), and 25 %mol of the indicated phospholipids. Data presented as mean ± SD of n - 3 biologically independent samples.

[0046] FIGS. 14A-14H show serine-substituted recombinant BMPS mutant exhibits weaker rescue of BMP depletion and LPG accumulation. FIG. 14A shows BMPS preferentially utilizes LPG for acyl donation. Recombinant BMPS (rCLN5) was incubated with different lysophospholipids and release of their corresponding glycerophosphodiester (GPD), BMP, and GPG was monitored. Data presented as mean ± SD of n = 3 biologically independent samples. FIGS. 14B-14E show targeted analyses of lysophosphatidylcholine (LPC), lysophosphatidylserine (LPS), lysophosphatidylethanolamine (LPE), and lysophosphatidylinositol (LPI) intensities. Fold changes in lipid species between CLN5 knockout and wildtype HEK293T lysosomes were calculated after subtracting background from control samples and normalizing to endogenous lipid. Data presented as mean ± SD of n = 4 biologically independent samples. FIG. 14F shows C231S rCLN5 activity monitored by BMP production. Recombinant BMPS WT and C231S were incubated with LPG (18:1) under acidic conditions. Data presented as mean ± SD of n = 3 biologically independent samples. ND, not detected. FIGS. 14G and 14H show C231S rCLN5 reduced rescue of LPG storage and BMP deficiency. Fold change in the levels of BMP and LPG species normalized to endogenous lipid. Data presented as mean ± SD of n = 4 biologically independent samples.

[0047] FIGS. 15A-15D show CLN5 deficient cells accumulate precursor phospholipid tracers. FIG. 15 A shows production of deuterated LPG in CLN5 deficient cells is not significantly affected. Intensities for d5-LPG production in CLN5 knockout and wildtype HEK293T cells are presented after background subtraction and normalization to endogenous lipids. Data presented as mean ± SD of n = 3 biologically independent samples. FIGS. 15B-15D show deuterated tracers accumulate in CLN5 deficient cells. Intensities for d5-PG (16:0/18:1), d9-PC (18:0/18:0), and d5-GlclCer (dl8: 1/18:0) tracers in CLN5 knockout and wildtype HEK293Ts are presented after background subtraction and normalization to endogenous lipids. Data presented as mean ± SD of n = 3 biologically independent samples.

[0048] FIGS. 16A and 16B show inhibition of cysteine protease increases BMP synthase protein levels. FIG. 16A demonstrates PG (18:1/18:1) supplementation and/or a cysteine protease inhibitor E.64 increase the levels of CLN5 protein. Data represents experiment repeated three times. FIG. 16B shows PG supplementation increases the levels of BMP species. Data represents experiment repeated three times.

[0049] FIGS. 17A-17C show glycerophosphodiesters bind BMPS. FIG. 17A demonstrates that GPG and glycerophosphatidylserine (GPS) thermally stabilize rBMPS. FIG. 17B demonstrates that GPS, glycerophosphatidylethanolamine (GPE), glycosylphosphatidylinositol (GPI), and glycerophosphatidylcholine (GPC) directly engage rBMPS at micromolar concentration as measured by MST. Data represents experiments repeated three times. FIG. 17C demonstrates that rBMPS does not exhibit GPD phosphodiesterase activity. Experiment repeated at least three times.

[0050] FIGS. 18A and 18B suggest that glycerophosphodiesters inhibit BMP synthase. FIG. 18A shows that GPG inhibits rBMPS. FIG. 18B demonstrates that all GPDs, except GPC, inhibit rBMPS. Experiment repeated at least three times.

[0051] FIGS. 19A-19D show that the interaction between glycerophosphodiesters and BMP synthase is mediated by the basic pocket. FIG. 19A demonstrates that GPG binds a basic pocket by docking one possible stereoisomer of GPG into a model of BMPS. FIG. 19B shows that this basic patch mediates direct binding between GPG and rBMPS. Experiment repeated at least three times. FIGS. 19C-D demonstrate that a GPD-null binding mutant of rBMPS is not inhibited by GPDs. FIG. 19C shows that rBMPS K296A retains BMP synthesis activity. FIG. 19D shows that rBMPS K296A is not inhibited by GPG. Experiment repeated at least three times

[0052] FIGS. 20A-20C show that BMPS N143S exhibits defective BMP synthesis activity. FIG. 20A shows that BMPS N143S exhibits attenuated BMPS synthesis activity. Recombinant BMPS WT and N143S were incubated with LPG (18:1) under acidic conditions. Data presented as mean ± SD of n = 3 biologically independent samples. FIGS. 20B and 20C show that BMPS N143S is unable to rescue LPG storage (FIG. 20C) and BMP deficiency (FIG. 20B). Fold change in the levels of BMP and LPG species normalized to endogenous lipid. Data presented as mean ± SD of n = 4 biologically independent samples.

[0053] FIGS. 21A-21F show BMPS mutants exhibit comparable uptake during enzyme replacement. FIG. 21A is a fluorescence image of fluorescent BMPS protein (Alexa488, green) and lysosomes (Lysotracker, red) of CLN5 knockout HEK293T cells. Scale bar = 5 pm. Representative image shown for experiment repeated at least three times. FIG. 21B shows recombinant BMPS WT and mutants localized to lysosomes but not endoplasmic reticulum (ER) or Golgi. Ten images were analyzed per condition for colocalization analysis. FIG. 21C is fluorescent images of fluorescent BMPS protein (Alexa488, green) and lysosomes (LAMP1- RFP, red). Scale bar = 2 pm. FIGS. 21D-21E are fluorescent images of fluorescent BMPS protein (Alexa488, green), endoplasmic reticulum (ERTracker, red), and Golgi apparatus (GolgiTracker, red) of CLN5 knockout HEK293T cells. Scale bar = 5 pm. FIG. 21F shows that recombinant BMPS N143S mutant exhibits no difference in cellular uptake compared to that of wildtype. Fluorescent BMPS intensities were determined and are presented relative to wildtype. Relative BMPS N143S uptake are compared to that of BMPS WT in two different experiments. Twenty cells were analyzed per condition for intensity measurements.

[0054] FIGS. 22A and 22B show alterations in hexosylceramide metabolism in BMPS deficient cells. CLN5 -deficient cells exhibit variable hexosylceramide metabolism. Fold changes in lipid species abundance between CLN5 knockout and wildtype HEK293T cells, iPSCs, and iNeurons were calculated after subtracting background from control samples and normalizing to endogenous lipid. Data presented as mean ± SD of n = 4 biologically independent samples for HEK293T cells and as mean ± SD of n = 3 biologically independent samples for iPSCs and iNeurons. [0055] FIG. 23 shows BMPS addition rescues Niemann-Pick Type C (NPC) disease phenotype in vitro. Fibroblasts derived from a healthy patient or NPC patient were treated with vehicle or BMPS and stained for filipin (left). Total filipin intensity per cell (right).

[0056] FIG. 24 shows that exogenous PGRN addition increases BMP synthesis in vitro. Intensities for d5-BMP (18:1/18:1) in Gm+/- mice knockout with and without exogenously provided PGRN-6XHis protein.

[0057] FIG. 25 shows exogenous BMPS treatment rescues defective GCase activity in Gm knockout cells. Intensities for LysoFQ-GBA in Gm knockout cells treated with vehicle, BMPS and loss-of-function BMPS mutant were normalized to vehicle treated Gm +/- BMDMs. ****, p<0.0001, by one way ANOVA.

[0058] FIG. 26 shows BMPS knockout cells exhibit defective GCase activity. Intensities for LysoFQ-GBA in BMPS knockout cells (Cln5 -/-) treated with vehicle, BMPS and loss-of- function BMPS mutant were normalized to vehicle treated Cln5 +/- BMDMs. ****, p<0.0001, by one way ANOVA.

[0059] FIG. 27 shows BMPS gene replacement restores after transient transfection BMP synthesis in BMPS knockout cells. Abundance of BMP was measured with triple quadrupole mass spectrometry in wild-type and BMPS knockout cells (Cln5 with vehicle and BMPS knockout cells transfected with an exogenous vector configured to express FLAG-tagged BMPS (KO + FLAG-C V5). Statistics were performed by one-way ANOVA in Graphpad, * p<0.05, ** p<0.01, ***p<0.001.

[0060] FIG. 28 shows characterization and validation of AAV-mediated gene replacement of the BMPS (CLN5) gene in CLN5 knock-out cells by western blot analysis of BMPS protein levels in wildtype HEK293T cells (WT), CLN5 knockout HEK293T cells (KO), and CLN5 knockout HEK293T cells treated with an AAV vector containing the gene for FLAG-tagged BMPS (KO+AAV). Treatment with AAV was able to successfully restore expression of BMPS protein.

[0061] FIG. 29 shows BMPS gene replacement after viral gene replacement restores BMP synthesis in BMPS knockout cells. Abundance of BMP was measured with triple quadrupole mass spectrometry in wild-type (WT) and BMPS knockout HEK293T cells (CLN5 labeled “KO”) with vehicle and BMPS knockout HEK293T cells treated with a FLAG-tagged BMPS AAV vector (KO + AAV-FLAG-CLN5). Statistics were performed by one-way ANOVA in Graphpad, * p<0.05, ** p<0.01, ***p<0.001.

DETAILED DESCRIPTION

[0062] As disclosed herein, the gene CLN5 was found to encode an enzyme capable of synthesizing bis(monoacylglycero)phosphate (BMP), henceforth known as BMP synthase (BMPS). BMPS knockout cells exhibited a massive accumulation of the BMP precursor lysophosphatidylglycerol (LPG), depletion of BMP species, and dysfunctional lipid metabolism. BMPS mediated synthesis through an energy-independent base exchange reaction between two LPG molecules with increased activity on BMP-laden vesicles. Provided herein are methods for modulating the level and/or the activity of BMPS. Particularly, methods are provided for increasing the level and/or activity of BMPS for stimulating and/or maintaining lysosomal function in a subject. For example, in a subject with a disease or disorder characterized by lysosomal dysfunction. Also provided are methods for decreasing the level or activity of BMPS, for example in diseases and disorders vulnerable to lysosome membrane permeabilization or infectious diseases which utilize lipids for cell entry.

[0063] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.

Definitions

[0064] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context.

[0065] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 arc explicitly contemplated.

[0066] As used herein, the modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” may refer to ±10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9 - 1.1. Other meanings of “about” may be apparent from the context, such as rounding off; for example, “about 1” may also mean from 0.5 to 1.4.

[0067] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0068] The term “amino acid” or “any amino acid” as used here refers to any and all amino acids, including naturally occurring amino acids (e.g., a-amino acids), unnatural amino acids, modified amino acids, and non-natural amino acids. It includes both D- and L-amino acids.

Natural amino acids include those found in nature, such as, e.g., the 23 amino acids that combine into peptide chains to form the building -blocks of a vast array of proteins. These arc primarily L stereoisomers, although a few D-amino acids occur in bacterial envelopes and some antibiotics. The “non-standard,” natural amino acids include, for example, pyrolysine (found in methanogenic organisms and other eukaryotes), selenocysteine (present in many non-eukaryotes as well as most eukaryotes), and N-formylmethionine (encoded by the start codon AUG in bacteria, mitochondria, and chloroplasts). “Unnatural” or “non-natural” amino acids are non- proteinogenic amino acids (e.g., those not naturally encoded or found in the genetic code) that either occur naturally or are chemically synthesized. Over 140 unnatural amino acids are known and thousands of more combinations are possible. Examples of “unnatural” amino acids include P-amino acids (P³ and p²), homo-amino acids, proline and pyruvic acid derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, D-amino acids, alpha-mcthyl amino acids and N-mcthyl amino acids. Unnatural or non-natural amino acids also include modified amino acids.

“Modified” amino acids include amino acids (e.g., natural amino acids) that have been chemically modified to include a group, groups, or chemical moiety not naturally present on the amino acid. According to certain embodiments, a peptide inhibitor comprises an intramolecular bond between two amino acid residues present in the peptide inhibitor. It is understood that the amino acid residues that form the bond will be altered somewhat when bonded to each other as compared to when not bonded to each other. Reference to a particular amino acid is meant to encompass that amino acid in both its unbonded and bonded state.

[0069] For the most part, the names of naturally occurring and non-naturally occurring aminoacyl residues used herein follow the naming conventions suggested by the IUPAC Commission on the Nomenclature of Organic Chemistry and the IUPAC-IUB Commission on Biochemical Nomenclature as set out in “Nomenclature of a- Amino Acids (Recommendations, 1974)” Biochemistry, 14(2), (1975). To the extent that the names and abbreviations of amino acids and aminoacyl residues employed in this specification and appended claims differ from those suggestions, they will be made clear to the reader.

[0070] Throughout the present specification, unless naturally occurring amino acids are referred to by their full name (e.g., alanine, arginine, etc.), they are designated by their conventional three-letter or single-letter abbreviations (e.g., Ala or A for alanine, Arg or R for arginine, etc.). The term “L-amino acid,” as used herein, refers to the “L” isomeric form of a peptide, and conversely the term “D-amino acid” refers to the “D” isomeric form of a peptide (e.g., Dphe, (D)Phe, D-Phe, or ^DF for the D isomeric form of Phenylalanine). Amino acid residues in the D isomeric form can be substituted for any L-amino acid residue, as long as the desired function is.

[0071] In the case of less common or non-naturally occurring amino acids, unless they are referred to by their full name (e.g. sarcosine, ornithine, etc.), frequently employed three- or four- character codes are employed for residues thereof, including, Sar or Sarc (sarcosine, i.e. N- methylglycine), Aib (a-aminoisobutyric acid), Dab (2,4-diaminobutanoic acid), Dapa (2,3- diaminopropanoic acid), y-Glu (y-glutamic acid), Gaba (y- aminobutanoic acid), -Pro (pyrrolidine-3-carboxylic acid), and 8Ado (8-amino-3,6-dioxaoctanoic acid), Abu (2-amino butyric acid), hPro (P-homoproline), hPhe (P-homophenylalanine) and Bip (3,3 diphcnylalaninc), and Ida (Iminodiacetic acid).

[0072] “Antibody” and “antibodies” as used herein refers to monoclonal antibodies, polyclonal antibodies, monospecific antibodies (e.g., which can either be monoclonal, or may also be produced by other means than producing them from a common germ cell), multi- specific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies such as, but not limited to, a bird (for example, a duck or a goose), a shark, a whale, and a mammal, including a non-primate (for example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, etc.) or a non-human primate (for example, a monkey, a chimpanzee, etc.), recombinant antibodies, chimeric antibodies, single-chain Fvs (“scFv”), single chain antibodies, single domain antibodies, Fab fragments, F(ab’) fragments, F(ab’)2 fragments, disulfide-linked Fvs (“sdFv”), and anti-idiotypic (“anti-Id”) antibodies, dual-domain antibodies, dual variable domain (DVD) or triple variable domain (TVD) antibodies (dual-variable domain immunoglobulins and methods for making them are described in Wu, C., et al., Nature Biotechnology, 25(11): 1290-1297 (2007) and PCT International Application WO 2001/058956, the contents of each of which are herein incorporated by reference), or domain antibodies (dAbs) (e.g., such as described in Holt et al., Trends in Biotechnology 21:484-490 (2014)), and including single domain antibodies sdAbs that are naturally occurring, e.g., as in cartilaginous fishes and camelid, or which are synthetic, e.g., nanobodies, VHH, or other domain structure), and functionally active epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, namely, molecules that contain an analyte-binding site. Immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA, and IgY), class (for example, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass.

[0073] Typically, an immunoglobulin or antibody is a protein that comprises at least one complementarity determining region (CDR). The CDRs form the “hypcrvariablc region” of an antibody, which is responsible for antigen binding. “CDR” is used herein to refer to the “complementarity determining region” within an antibody variable sequence. There are three CDRs in each of the variable regions of the heavy chain and the light chain. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted “CDR1,” “CDR2,” and “CDR3,” for each of the variable regions. The term “CDR set” as used herein refers to a group of three CDRs that occur in a single variable region that binds the antigen. An antigen-binding site, therefore, may include six CDRs, comprising the CDR set from each of a heavy and a light chain variable region. A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2, or CDR3) may be referred to as a “molecular recognition unit.” Crystallographic analyses of antigen- antibody complexes have demonstrated that the amino acid residues of CDRs form extensive contact with bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Thus, the molecular recognition units may be primarily responsible for the specificity of an antigenbinding site. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

[0074] A whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (VH) region and three C-terminal constant (CHI, CH2, and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The light chains of antibodies can be assigned to one of two distinct types, either kappa (K) or lambda (/.), based upon the amino acid sequences of their constant domains. In a typical antibody, each light chain is linked to a heavy chain by disulfide bonds, and the two heavy chains are linked to each other by disulfide bonds. The light chain variable region is aligned with the variable region of the heavy chain, and the light chain constant region is aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chains are aligned with each other.

[0075] The variable regions of each pair of light and heavy chains form the antigen binding site of an antibody. The VH and VL regions have the same general structure, with each region comprising four framework (FW or FR) regions. The term “framework region,” as used herein, refers to the relatively conserved amino acid sequences within the variable region which are located between the CDRs. There are four framework regions in each variable domain, which are designated FR1, FR2, FR3, and FR4. The framework regions form the sheets that provide the structural framework of the variable region (see, e.g., C. A. Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, N.Y. (2001)).

[0076] Humanized” forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypcrvariablc region of the recipient arc replaced by residues from a hypcrvariablc region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired antibody specificity, affinity, and capability. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a nonhuman immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0077] The term “monoclonal antibody,” as used herein, refers to an antibody produced by a single clone of B lymphocytes that is directed against a single epitope on an antigen. Monoclonal antibodies typically are produced using hybridoma technology, as first described in Kohler and Milstein, Eur. J. Immunol., 5: 511-519 (1976). Monoclonal antibodies may also be produced using recombinant DNA methods (see, e.g., U.S. Patent 4,816,567), isolated from phage display antibody libraries (see, e.g., Clackson et al. Nature, 352: 624-628 (1991)); and Marks et al., J. Mol. Biol., 222: 581-597 (1991)), or produced from transgenic mice carrying a fully human immunoglobulin system (see, e.g., Lonberg, Nat. Biotechnol., 23(9): 1117-25 (2005), and Lonberg, Handb. Exp. Pharmacol., 181: 69-97 (2008)). In contrast, “polyclonal” antibodies are antibodies that are secreted by different B cell lineages within an animal. Polyclonal antibodies are a collection of immunoglobulin molecules that recognize multiple epitopes on the same antigen.

[0078] The term “monospecific” antibody as used herein denotes an antibody that has one or more binding sites each of which bind to the same epitope of the same antigen.

[0079] The term “bispecific” antibody as used herein denotes an antibody that has at least two binding sites each of which bind to different epitopes of the same antigen or a different antigen. [0080] The term “multi-specific” antibody as used herein denotes an antibody that has binding specificities for at least two different sites.

[0081] As used herein, a “nucleic acid” or a “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and/or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793- 800 (Worth Pub. 1982)). The present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single- stranded or double- stranded form, including homoduplex, heteroduplex, and hybrid states. The term “nucleic acid” or “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and/or non- nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”); further, the term “nucleic acid sequence” as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or double-stranded, and represent the sense or antisense strand. The terms “nucleic acid,” “polynucleotide,” “nucleotide sequence,” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.

[0082] As used herein, the term “percent sequence identity” refers to the percentage of nucleotides or nucleotide analogs in a nucleic acid sequence, or amino acids in an amino acid sequence, that is identical with the corresponding nucleotides or amino acids in a reference sequence of the present disclosure after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Hence, in case a nucleic acid or protein is longer than a reference sequence, additional nucleotides or amino acids that do not align with the reference sequence are not taken into account for determining sequence identity. A number of mathematical algorithms for obtaining the optimal alignment and calculating identity between two or more sequences are known and incorporated into a number of available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1 , BL2SEQ, and later versions thereof) and FASTA programs (c.g., FASTA3x, FAS™, and SSEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms also are disclosed in, for example, Altschul et al., J. Molecular Biol., 215(3): 403-410 (1990), Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10): 3770-3775 (2009), Durbin et al., eds., Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009), Soding, Bioinformatics, 21(7): 951-960 (2005), Altschul et al., Nucleic Acids Res., 25(17): 3389-3402 (1997), and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)).

[0083] A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. The peptide or polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. Polypeptides include proteins such as binding proteins, receptors, and antibodies. The polypeptides may be modified by the addition of sugars, lipids or other moieties not included in the amino acid chain.

[0084] An “effective amount” refers to an amount sufficient to elicit a desired biological response (e.g., treating a condition). As will be appreciated by those skilled in the art, the effective amount may vary depending on such factors as the desired biological endpoint, the pharmacokinetics, the condition being treated, the mode of administration, and the age and health of the subject. An effective amount encompasses therapeutic and prophylactic treatment. For example, a “therapeutically effective amount” is an amount sufficient to provide a therapeutic benefit in the treatment of a condition, or to delay or minimize one or more symptoms associated with the condition. In some embodiments, a therapeutically effective amount is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to minimize one or more symptoms associated with the condition. A therapeutically effective amount means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the condition, or enhances the therapeutic efficacy of another therapeutic agent.

[0085] A “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein. Likewise, subject may include either adults or juveniles (e.g., children). Moreover, subject may mean any living organism, preferably a mammal (e.g., humans and nonhumans) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment, the mammal is a human.

[0086] A cell has been “genetically modified,” “transformed,” or “transfected” by exogenous DNA, e.g., a recombinant expression vector, when such DNA has been introduced inside the cell. The presence of the exogenous DNA results in permanent or transient genetic change. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. For example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones that comprise a population of daughter cells containing the transforming DNA. A “clone” is a population of cells derived from a single cell or common ancestor by mitosis. A “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.

[0087] A “vector” or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, e.g., an “insert,” may be attached or incorporated so as to bring about the replication of the attached segment in a cell.

[0088] As used herein, the terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or condition, or one or more signs or symptoms thereof. In some embodiments, “treatment,” “treat,” and “treating” require that signs or symptoms of the disease disorder or condition have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease or condition. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.

[0089] As used herein, the terms “providing,” “administering,” and “introducing,” are used interchangeably herein and refer to the placement of the disclosed proteins, polypeptides, nucleic acids, polynucleotides, and small molecules into a cell, organism, or subject by a method or route which results in at least partial localization to a desired site. The administration can be by any appropriate route which results in delivery to a desired location in the cell, organism, or subject. [0090] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75^th Ed., inside cover, and specific functional groups are generally defined as described therein.

Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Sorrell, Organic Chemistry, 2^nd edition, University Science Books, Sausalito, 2006; Smith, March's Advanced Organic Chemistry: Reactions, Mechanism, and Structure, 7^th Edition, John Wiley & Sons, Inc., New York, 2013; Larock, Comprehensive Organic Transformations, 3^rd Edition, John Wiley & Sons, Inc., New York, 2018; and Carruthers, Some Modern Methods of Organic Synthesis, 3^rd Edition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0091] As used herein, “bis(monoacylglycero)phosphate” or “BMP” refers to a compound having the general formula: wherein R and R’ are independently each selected from C4-C4oalkyl, C4-C4oalkenyl, or C4-C4oalkynyl, as defined herein.

[0092] As used herein, “bis(monoacylglycero)phosphate synthase,” “BMP synthase,” or “BMPS” refers to any polypeptide comprising the enzymatic functionality to produce BMP, e.g., from lysophospholipid substrates. Herein, BMPS is also referred to as CLN5 protein, or the protein encoded by CLN5.

[0093] As used herein, “glycerophosphocholine” or “GPC” refers to a compound having the general formula including all stereoisomers of:

[0094] As used herein, “glycerophosphoethanolamine” or “GPE” refers to a compound having the general formula including all stereoisomers of:

[0095] As used herein, “glycerophosphoglycerol” or “GPG” refers to the glycerol ester of glycerophosphoric acid having the general formula including all stereoisomers of: [0096] As used herein, “glycerophosphoinositol” or “GPI” refers to a compound having the general formula including all stereoisomers of:

[0097] As used herein, “glycerophospho serine” or “GPS” refers to a compound having the general formula including all stereoisomers of:

[0098] “Lysophospholipid” refers to small lipid molecules characterized by a single carbon chain and a polar head group. Lysophospholipid subgroups can be distinguished by their backbones. For example, lysophospholipids containing the sphingoid base backbone are lysosphingolipids, whereas lysophospholipids containing the glycerol backbone are lysoglycerophospholipids. Exemplary lysophospholipids, include, without limitation, lysophosphatidylcholine (LPC), lysophosphatidylserine (LPS), lysophosphatidylethanolamine (LPE), lysophosphatidylglycerol (LPG), lysophosphatidic acid (LPA), 2,3-cyclic phosphatidic acid, l-alkyl-2-acetyl-glycero-3-phosphate, sphingosine- 1 -phosphate (SIP), dihydro- sphingosinc-1 -phosphate, sphingosylphosphorylcholinc (lysosphingomyclin, SPC), and lysophosphatidylinositol (LPI).

[0099] As used herein, “lysophosphatidylglycerol” or “LPG” refers to an ester phospholipid having the general formulas

, in R is a C4-C4o lkyl, C4-C4o lkenyl, or C4-C4o lkynyl, as defined herein.

[00100] As used herein, “phosphatidylglycerol” or “PG” refers to a compound having the general formula: , wherein R and R’ are independently each selected from

C4-C4oalkyl, C4-C4oalkenyl, or C4-C4oalkynyl, as defined herein.

[00101] The term “alkyl,” as used herein, means a straight or branched, saturated hydrocarbon chain. For example, C4-C4oalkyl means a straight or branched, saturated hydrocarbon chain containing from 4 to 40 carbon atoms (e.g., 4 to 20 carbons, 10 to 40 carbons, 10 to 30 carbons, 10 to 20 carbons, or 20 to 30 carbons). Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec -butyl, iso-butyl, tent-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 4,4- dimethylpentan-2-yl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

[00102] The term “alkenyl,” as used herein, means a straight or branched, hydrocarbon chain containing at least one carbon-carbon double bond. [00103] The term “alkynyl,” as used herein, means a straight or branched, hydrocarbon chain containing at least one carbon-carbon triple bond.

[00104] In some instances, the number of carbon atoms in a hydrocarbyl substituent (e.g., alkyl alkenyl) is indicated by the prefix “C_x-C_y”, wherein x is the minimum and y is the maximum number of carbon atoms in the substituent. Thus, for example, “C1-C3 alkyl” refers to an alkyl substituent containing from 1 to 3 carbon atoms.

[00105] For compounds described herein, groups and substituents thereof may be selected in accordance with permitted valence of the atoms and the substituents, such that the selections and substitutions result in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

[00106] Where substituent groups are specified by their conventional chemical formulae, written from left to right, they optionally encompass substituents resulting from writing the structure from right to left, e.g., -CH2O- is intended to encompass -OCH2-, and -C(O)NH- is intended to encompass -NHC(O)-.

[00107] Any of the described compounds may exist as a stereoisomer wherein asymmetric or chiral centers are present. The stereoisomer is “R” or “S” depending on the configuration of substituents around the chiral carbon atom. The terms “R” and “S” used herein are configurations as defined in IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry, in Pure Appl. Chem., 1976, 45: 13-30. The disclosure contemplates various stereoisomers and mixtures thereof of the described compounds. Stereoisomers include enantiomers and diastereomers, and mixtures of enantiomers or diastereomers. It should be understood that the described compounds may possess tautomeric forms, as well as geometric isomers, and that these also constitute embodiments of the disclosure.

[00108] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

Methods for Increasing the Level or Activity of BMPS

[00109] Provided herein are methods for stimulating and/or maintaining lysosomal function in a subject. The method comprises increasing the level and/or activity of bis(monoacylglycero)phosphate (BMP) synthase in the subject. In certain embodiments, the level and/or activity of BMP synthase is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more as compared to a control which does not undergo the disclosed methods. In some embodiments, the level and/or activity of BMP synthase is increased by at least 1-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. Measuring levels of a protein are well known to one of skill in the art. The activity of BMP synthase may be measured by methods as disclosed herein. For example, by quantifying BMP and byproducts. [00110] In some embodiments, increasing the level and/or activity of BMP synthase may be by administering to the subject an effective amount of a BMP synthase activator or a composition thereof.

[00111] The activator may be any agent which increases the level and/or activity of BMP synthase. For example, the activator may be a small molecule activator of BMP synthase, progranulin, granulin peptides, phosphatidylglycerol or derivatives thereof, lysophosphatidylglycerol or derivatives thereof, or combinations thereof.

[00112] In some embodiments, the activator comprises a small molecule activator of BMP synthase. In some embodiments, the activator comprises progranulin, granulin peptides, or derivatives thereof (e.g., human granulin peptides A, B, C, D, E, F, and G). For example, see also Boland et al., bioRxiv 2021.09.30.461806; doi.org/10.1101/2021.09.30.461806, incorporated herein by reference. In some embodiments, the activator comprises phosphatidylglycerol or derivatives thereof, lysophosphatidylglycerol or derivatives thereof, or combinations thereof. In some embodiments, the activator comprises a protease inhibitor (e.g., a cysteine protease inhibitor).

[00113] In some embodiments, increasing the level and/or activity of BMP synthase may be increased by administering to the subject an effective amount of a BMP synthase, a catalytically active fragment or variant thereof, or a nucleic acid encoding BMP synthase or a catalytically active fragment or variant thereof. BMP synthase, as used herein, refers to any enzyme of fragment thereof which catalyzes acylation of lysophosphatidylglycerol (LPG). In some embodiments, the BMP synthase comprises an amino acid sequence of SEQ ID NO: 1. In some embodiments, the BMP synthase comprises an amino acid sequence of SEQ ID NO: 2. In some embodiments, the BMP synthase comprises an amino acid sequence at least 70% similar to residues 115-240, relative to SEQ ID NO: 1 . In some embodiments, the BMP synthase comprises an amino acid sequence of residues 115-240, relative to SEQ ID NO: 1.

[00114] Variants of BMP synthase useful for the methods disclosed herein may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or more) amino acid substitutions as compared to a native or wild-type BMP synthase or SEQ ID NOs: 1 or 2 while retain catalytic (e.g., BMP synthesis) activity. An amino acid “replacement” or “substitution” refers to the replacement of one amino acid at a given position or residue by another amino acid at the same position or residue within a polypeptide sequence. Amino acids are broadly grouped as “aromatic” or “aliphatic.” An aromatic amino acid includes an aromatic ring. Examples of “aromatic” amino acids include histidine (H or His), phenylalanine (F or Phe), tyrosine (Y or Tyr), and tryptophan (W or Trp). Non- aromatic amino acids are broadly grouped as “aliphatic.” Examples of “aliphatic” amino acids include glycine (G or Gly), alanine (A or Ala), valine (V or Vai), leucine (L or Leu), isoleucine (I or He ), methionine (M or Met), serine (S or Ser), threonine (T or Thr), cysteine (C or Cys), proline (P or Pro), glutamic acid (E or Glu), aspartic acid (A or Asp), asparagine (N or Asn), glutamine (Q or Gin), lysine (K or Lys), and arginine (R or Arg).

[00115] The amino acid replacement or substitution can be conservative, semi-conservative, or non-conservative. The phrase “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz and Schirmer, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz and Schirmer, supra). Examples of conservative amino acid substitutions include substitutions of amino acids within the sub-groups described above, for example, lysine for arginine and vice versa such that a positive charge may be maintained, glutamic acid for aspartic acid and vice versa such that a negative charge may be maintained, serine for threonine such that a free -OH can be maintained, and glutamine for asparagine such that a free -NH2 can be maintained. “Semi-conservative mutations” include amino acid substitutions of amino acids within the same groups listed above, but not within the same sub-group. For example, the substitution of aspartic acid for asparagine, or asparagine for lysine, involves amino acids within the same group, but different sub-groups. “Non-conservative mutations” involve amino acid substitutions between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc.

[00116] As such, in some embodiments, the BMP synthase comprises an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%) identity to SEQ ID NO: 1. In some embodiments, the BMP synthase comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 1. [00117] As such, in some embodiments, the BMP synthase comprises an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%) identity to SEQ ID NO: 2. In some embodiments, the BMP synthase comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 2. [00118] In some embodiments, the BMP synthase, the catalytically active fragment or variant thereof, or BMP synthase activator is linked to a polypeptide or molecule configured to bind to a blood-brain barrier (BBB) receptor (e.g., a receptor associated protein (RAP)). For example, the BMP synthase or BMP synthase activator may be linked to molecules such as endogenous ligands or monoclonal antibodies that act to bind exofacial epitopes on BBB receptor-mediated transport systems, triggering internalization of the receptor and of the BMP synthase. In some embodiments, the BMP synthase or BMP synthase activator is linked to a polypeptide comprising a receptor-binding domain from an apolipoprotein, or an engineered variant thereof. A receptor binding domain of an apolipoprotein, for example, can be chosen from the receptor binding domain of ApoA, ApoB, ApoC, ApoD, ApoE, ApoE2, ApoE3, ApoE4, and combinations thereof. In some embodiments, the BMP synthase or BMP synthase activator is linked to a polypeptide comprising a transferrin-receptor binding site.

[00119] In some embodiments, the BMP synthase or BMP synthase activator is linked to a polypeptide comprising a first Fc region. Accordingly, the disclosure provides a fusion protein comprising a BMP synthase and a polypeptide comprising a first Fc region. A fusion protein comprising a BMP synthase and an Fc domain (BMPS-Fc fusion protein) refers to a fusion protein in which a BMP synthase is linked, directly or indirectly, to a polypeptide comprising a first Fc region. Use of this fusion protein may facilitate brain penetration. [00120] The BMP synthase and the polypeptide comprising a first Fc region may be linked in any orientation. In some embodiments, the N-tcrminus of the BMP synthase is linked to the C- terminus of the polypeptide comprising a first Fc region. In some embodiments, the C-terminus of the BMP synthase is linked to the N-terminus of the polypeptide comprising a first Fc region. In some embodiments, the N-terminus of the BMP synthase is linked to the N-terminus of the polypeptide comprising a first Fc region. In some embodiments, the C-terminus of the BMP synthase is linked to the C-terminus of the polypeptide comprising a first Fc region.

[00121] In some embodiments, the BMP synthase-Fc fusion protein comprises a linker between the BMP synthase and the polypeptide comprising a first Fc region. The linker may have any of a variety of amino acid sequences and be a variety of lengths (e.g., 4-100 amino acids). The linker can be produced by using synthetic, linker-encoding oligonucleotides to couple the portions of the fusion protein or can be encoded by a nucleic acid sequence encoding the fusion protein. In some embodiments, the linker polypeptide is considered a flexible linker, facilitating some degree of orientation freedom for BMP synthase and the polypeptide comprising a first Fc region. A variety of different linkers are considered suitable for use, including but not limited to, glycine- serine polymers, glycine-alanine polymers, and alanineserine polymers.

[00122] In some embodiments, the polypeptide comprising a first Fc region is an antibody. In some embodiments, the antibody is a monoclonal antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a monospecific antibody, a bispecific antibody, or a multispecific antibody. In some embodiments, the antibody or fragment thereof is configured to bind to a blood-brain barrier (BBB) receptor, e.g., antibodies that bind endothelial cell receptors resulting in endocytosis of the receptor and bound ligands. See for example U.S. Patent No. 7,744,879 and U.S. Patent Application Publication No. 2016/0369001, each incorporated herein by reference in their entirety.

[00123] In some embodiments, the methods further comprise administering a polypeptide comprising a second Fc region configured to form a dimer with the first Fc region.

[00124] In some embodiments, the polypeptide comprising a second Fc region be linked to a BMP synthase activator or a BMP synthase, a catalytically active fragment or variant thereof. Thus, each of the polypeptides comprising the first Fc region and second Fc region are delivering a cargo which results individually or in combination with the other to increase the level or activity of BMP synthase.

[00125] In some embodiments, the polypeptide comprising a first Fc region and/or the polypeptide comprising a second Fc region binds to a blood-brain barrier (BBB) receptor, e.g., endothelial cell receptors resulting in endocytosis of the receptor and bound ligands. In some embodiments, the polypeptide comprising a first Fc region and/or the polypeptide comprising a second Fc region binds to a transferrin receptor.

[00126] The terms “Fc domain,” “Fc,” or “Fc region” are used interchangeably herein and refer to the polypeptide comprising the constant region of an antibody excluding, in some instances, the first constant region immunoglobulin domain (e.g., CHI) or a portion thereof, and in some cases, part of the hinge. Thus, an Fc domain can refer to the last two constant region immunoglobulin domains (e.g., CH2 and CH3) of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM, Fc may include the J chain. In some embodiments, an Fc domain refers to a truncated CHI domain, and CH2 and CH3 of an immunoglobulin. Although the boundaries of the Fc domain may vary, the human IgG heavy chain Fc domain is usually defined to include residues E216 or C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat. However, the C-terminal lysine (Lys447) of the Fc domain may or may not be present, without affecting the structure or stability of the Fc domain. [00127] In certain embodiments, Fc domain refers to an immunoglobulin IgG heavy chain constant region comprising a hinge region (starting at Cys226), an IgG CH2 domain and CH3 domain. The term “hinge region” or “hinge sequence” as used herein refers to the amino acid sequence located between the linker and the CH2 domain. Fc region from an IgG subclass of any given animals. For example, in humans, the IgG classes including IgGl, IgG2, IgG3, and IgG4; in mouse the IgG classes including IgGl, IgG2a, IgG2b, and IgG3; and in rat the IgG classes including IgGl, IgG2a, IgG2b, IgG2c, and IgG3. It is known that certain IgG subclasses, for example, rat IgG2b and IgG2c, have higher clearance rates than, for example, IgGl. Thus, when using IgG subclasses other than IgGl it may be advantageous to substitute one or more of the residues, particularly in the CH2 and CH3 domains, which differ from the IgGl sequence with those of IgGl, thereby increasing the in vivo half-life of the other types of IgG. In certain particular embodiments, the Fc domain comprises the Fc domain of human IgGl, IgG2, IgG3 or IgG4. In certain other particular embodiments, the Fc domain comprises the CH2 and CH3 domain of IgG 1.

[00128] In some embodiments, the Fc domain is a native sequence Fc domain. In some embodiments, amino acid modifications are made to the Fc domain, for example to alter binding to one or more receptors or to alter serum half-life, by modifying or engineering the native sequence Fc domain. The possible variants of altered Fc-fusion proteins useful with the present invention are many and range from the changing of just one or a few amino acids to the complete redesign of, for example, the constant region. Changes in the constant region will, in general, be made in order to improve, or alter (e.g., increase or decrease) characteristics, such as binding interactions with various Fc-gamma receptors and/or other immunoglobulin effector functions. In some embodiments, an Fc domain is altered to increase or decrease the extent to which the fusion protein is glycosylated. Addition or deletion of glycosylation sites to a protein may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed. In certain embodiments, one or more residues of the Fc domain are substituted with cysteine residues, to position reactive thiol groups at accessible sites of the Fc domain, for use in conjugating the Fc domain to other moieties. In some embodiments, the first Fc region and the second Fc region comprise modifications that promote heterodimerization .

[00129] The BMP synthase or BMP synthase activator may further comprise a lysosomal targeting moiety, which once administered, will target the BMP synthase to the lysosome. Exemplary lysosomal targeting domains may be derived from peptides including, but not limited to, IGF-II, IGF-I, Kif, ApoE, TAT, RAP, and p97 peptide.

[00130] The BMP synthase or BMP synthase activator may further comprise one or more lipid moieties. Lipids which can be covalently attached to proteins include, for example, fatty acids, isoprenoids, sterols, and phospholipids. The lipid moieties may be directly or indirectly attached to any part of the BMP synthase. The attachment of the lipid moieties may improve the half-life of the BMP synthase and increase its ability to penetrate the blood-brain barrier, and thus increase its overall efficacy as compared to a BMP synthase lacking the lipid moiety. [00131] The BMP synthase may further comprise an epitope tag (e.g., 3xFLAG tag, a polyhistidine tag, an HA tag, a Myc tag, and the like). The epitope tags may be at the N-terminus, a C-terminus, or a combination thereof of the corresponding protein. [00132] In some embodiments, the BMP synthase may be fused with one or more (e.g., two, three, four, or more) protein transduction domains or PTDs, also known as a CPP, cell penetrating peptide. A protein transduction domains is a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule, facilitates the molecule traversing a membrane, for example going from extracellular space to intracellular space, or cytosol to within an organelle. In some embodiments, a PTD is covalently linked to a terminus of the BMP synthase (e.g., N-terminus, C-terminus, or both). In some embodiments, the PTD is inserted internally at a suitable insertion site. Examples of PTDs include but are not limited to a minimal undecapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT comprising); a polyargininc sequence comprising a number of arginine residues sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginine residues); a VP22 domain; a Drosophila Antennapedia protein transduction domain; a truncated human calcitonin peptide; polylysine; transportan, and the like. [00133] In some embodiments, increasing the level and/or activity of BMP synthase may be by administering to the subject an effective amount of a BMP synthase activator or a composition thereof, as described above, and a BMP synthase, a catalytically active fragment or variant thereof, as described above, or a nucleic acid encoding BMP synthase or a catalytically active fragment or variant thereof. The administration of a BMP synthase activator and the administration of a BMP synthase, or nucleic acid encoding thereof, may be in a single formulation or in two different formulations administered at the same time. Alternatively, the administrations can be sequential, in either order, and separated by a period of time ranging from hours to months.

[00134] In some embodiments, the subject has or is suspected of having a disease or disorder characterized by lysosomal dysfunction, decreased lipid catabolism, and/or increased cholesterol accumulation. Accordingly, in some embodiments, disclosed herein are methods of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of a BMP synthase activator or a composition thereof and a BMP synthase, a catalytically active fragment or variant thereof, or a nucleic acid encoding BMP synthase or a catalytically active fragment or variant thereof. [00135] In certain embodiments, treatment according to the present disclosure results in a reduction (e.g., about a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 97.5%, 99% or more reduction) or a complete elimination of the presence, or alternatively the accumulation, of one or more pathological, clinical, or biological markers that are associated with the particular disease or disorder

[00136] In some embodiments, treatment refers to increased survival (e.g., survival time). For example, treatment can result in an increased life expectancy of a patient. In some embodiments, treatment results in an increased life survival by more than about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 195%, about 200% or more, as compared to an average survival time (e.g., life expectancy of one or more control individuals with a select disease without treatment). In some embodiments, treatment results in an increased life expectancy of a patient by more than about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years or more, as compared to the average life expectancy of one or more control individuals without treatment. In some embodiments, treatment results in long term survival of a patient. As used herein, the term “long term survival” refers to a survival time or life expectancy longer than about 40 year’s, 45 years, 50 years, 55 year’s, 60 years, or longer.

[00137] The term “improve,” “increase,” or “reduce,” as used herein, indicates values that are relative to a control. In some embodiments, a suitable control is a baseline measurement, such as a measurement in the same cell or same individual prior to initiation of the treatment described herein, or a measurement in a control cell(s) or individual(s) in the absence of the treatment described herein. For example, a “control individual” is an individual afflicted with a select disease, who is approximately the same age and/or gender as the individual being treated (to approximate that the stages of the disease in the treated individual and the control individual(s) are comparable). [00138] In some embodiments, the subject has or is suspected of having a neurodegenerative disease. A “neurodegenerative disease” or a “NDD” refers to a central nervous system disease characterized by progressive, normally gradual, loss of functional neural tissue. Non-limiting examples of neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Friedreich's ataxia, Multiple sclerosis, Huntington's disease, transmissible spongiform encephalopathy, Charcot-Marie-Tooth disease, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy, Bell’s palsy, neuronal ceroid lipofuscinoses, and hereditary spastic paraparesis. In select embodiments, the neurodegenerative disease is selected from Niemann-Pick disease, frontotemporal dementia (FTD), Parkinson’s disease (PD), Alzheimer’s disease (AD), a neuronal ceroid lipofuscinosis, amyotrophic lateral sclerosis (ALS), and dementia with Lewy bodies (DLB).

[00139] Neuronal ceroid lipofuscinoses are a family of a family of neurodegenerative lysosomal storage disorders characterized by the accumulation of storage material or ceroid in the lysosome and caused by mutations in one of at least thirteen related genes, including for example, ATP13A2, CLN3, CLN5, CLN6, CLN8, CTSD. CTSF, CTSK, DNAJC5, GRN, KCTD7, MFSD8, PANK2, PPT1, SGSH, and TPP1. All these disorders affect the nervous system and typically cause worsening problems with vision, movement, and thinking or cognitive ability. Each disease type is given the designation “CLN,” meaning ceroid lipofuscinosis, neuronal, and then a number to indicate its type. In some embodiments, the neuronal ceroid lipofuscinosis is selected from neuronal ceroid lipofuscinosis type 1 (CLN1), neuronal ceroid lipofuscinosis type 2 (CLN2), neuronal ceroid lipofuscinosis type 3 (CLN3), neuronal ceroid lipofuscinosis type 4 (CLN4), neuronal ceroid lipofuscinosis type 5 (CLN5), neuronal ceroid lipofuscinosis type 6 (CLN6), neuronal ceroid lipofuscinosis type 7 (CLN7), neuronal ceroid lipofuscinosis type 8 (CLN8), neuronal ceroid lipofuscinosis type 9 (CLN9), neuronal ceroid lipofuscinosis type 10 (CLN 10), neuronal ceroid lipofuscinosis type I I (CLN 11), neuronal ceroid lipofuscinosis type 12 (CLN12), neuronal ceroid lipofuscinosis type 13 (CLN13), and neuronal ceroid lipofuscinosis type 14 (CLN 14).

[00140] In some embodiments, the subject has or is suspected of having a lysosomal storage disorder. Lysosomal storage disorders (LSDs) are a family of over 70 types of inherited diseases characterized by lysosomal dysfunction and often showing a neurodegenerative course. LSDs applicable to the present invention include, but are not limited to, lipid storage disorders (e.g., sphingolipidoses, gangliosidoses, leukodystrophies), mucopolysaccharidoses, glycoprotein storage disorders, and mucolipidoses. Not limiting examples of lysosomal storage disorders include: Farber disease, Krabbe disease, galactosialidosis, Fabry disease, Schindler disease, GM1 gangliosidosis, GM2 gangliosidosis (e.g., Sandhoff disease, Tay-Sachs, GM2 activator deficiency), Gaucher disease, lysosomal acid lipase deficiency, Niemann-Pick diseases, metachromatic leukodystrophy, multiple sulfatase deficiency, Type I mucopolysaccharidoses (e.g., Hurler syndrome, Scheie syndrome, Hurler-Scheie syndrome), Type II mucopolysaccharidoses (e.g., Hunter syndrome), Type III mucopolysaccharidoses (e.g., Sanfilippo syndrome), Type IV mucopolysaccharidoses (e.g., Morquio syndrome), Type VI mucopolysaccharidoses (e.g., Maroteaux-Lamy syndrome), Type VII mucopolysaccharidoses (e.g., Sly syndrome), Type IX mucopolysaccharidoses (e.g., hyaluronidase deficiency), Type I- IV mucolipidosis (e.g., sialidosis, Pseudo-Hurler-Polydystrophy, I-cell disease), Wolman disease, Pompe disease, Danon disease, lysosomal transport disease (e.g., cystinosis, pycnodysostosis, Salla disease, infantile free sialic acid storage disease), metachromatic leukodystrophy, oligosaccharidosis, and sialuria. In select embodiments, the lysosomal storage disease is selected from: Gaucher’s disease, metachromatic leukodystrophy, GM1 gangliosidosis, Tay-Sachs disease, Sandhoff disease, Fabry disease, Farber disease, Wolman disease, mucolipidosis Types I, II/III and IV, Type I mucopolysaccharidoses, Type II mucopolysaccharidoses, Type III mucopolysaccharidoses, Type IV mucopolysaccharidoses, Type VI mucopolysaccharidoses, and Krabbe disease.

[00141] In some embodiments, the subject has or is suspected of having drug-induced phospholipidosis, familial hypercholesterolemia (FH), a cardiovascular disease (e.g., hypertension, ischemia, reperfusion injury including post-MI ischemic reperfusion injury, stroke including ischemic stroke, transient ischemic attack, myocardial infarction including recurrent myocardial infarction, heart failure including congestive heart failure and heart failure with preserved ejection fraction, embolism, aneurysms including abdominal aortic aneurysm, and pericarditis including Dressier’s syndrome, obesity), atherosclerosis, a fatty liver disease (e.g., non-alcoholic fatty liver disease (NAFLD) (e.g., non-alcoholic steatohepatitis) and alcoholic liver disease), or any combination thereof.

[00142] In some embodiments, the methods may further comprise administration with one or more additional therapies to treat the disease or disorder, or one or more symptoms of the disease or disorder. The additional therapy may include administration of an additional therapeutic agent or a therapy not connected to administration of another agent including surgery and physical, occupational, and/or speech therapy.

[00143] The additional therapy may be administered at the same time as the initial therapy. For example, either in the same composition or in a separate composition administered at substantially the same time as the first composition. In some embodiments, the additional therapy may precede or follow the treatment of the initial therapy by time intervals ranging from hours to months.

[00144] The methods may further comprise activating autophagy or autophagy related pathways, activating the function or enhancing the levels of transcription factor EB (TFEB), or a combination thereof. Autophagy modulators including small-molecule PPARa agonists, such as fenofibrate, bezafibrate and gemfibrozil, mitigate of autophagy deficits. Gemfibrozil decreases cellular accumulates, improves motor coordination and increased longevity. PPARa is known to enhance levels of transcription factor EB (TFEB), which subsequently binds to promoters of genes involved in lysosome biogenesis, increasing their expression. TFEB translocation to the nucleus can be targeted therapeutically by inhibition of protein kinase B (AKT). Transport of TFEB to the nucleus is regulated by AKT-driven phosphorylation of TFEB, and inhibition of that phosphorylation results in increased nuclear TFEB and activation of the coordinated lysosomal expression and regulation (CLEAR) signaling network. TFEB activation enhances clearance of aggregates, improves behavior, and increases longevity. In some embodiments, TFEB, a catalytically active fragment or variant thereof, or a nucleic acid encoding TFEB or a catalytically active fragment or variant thereof may be administered. See for example, U.S. Patent Publications 2022/0185854 and 2014/0038897, incorporated herein by reference in their entirety. Targeting autophagy and/or TFEB may also be accomplished through other pathways such as the PI3K-mT0R pathway or AMP-activated protein kinase.

[00145] The methods may further comprise treatment with immune modulators and/or neuroprotective compounds. Neuroinflammation has been shown to exacerbate neurodegeneration. Immunotherapies have been explored in a number of neurodegenerative diseases, including PD, ALS, and AD, with preclinical success. Immunomodulation with mycophenolate mofetil improved motor coordination, reduced levels of serum autoantibodies and reduced neuroinflammation. Fingolimod, which impairs lymphocyte emigration into the brain via sphingosine- 1 -phosphate receptor modulation, and teriflunomide, which reduces the proliferation of activated immune cells via pyrimidine nucleotide synthesis inhibition, reduce neuron loss, brain atrophy and retinal thinning. Other potential immune modulators and neuroprotective compounds include, but are not limited to, steroids (e.g., prednisolone, allopregnanolone, vamorolone), phosphodiesterase 4 inhibitors (e.g., rolipram, roflumilast, PF- 06266047), cannabinoids, ol receptor agonists, excitotoxicity and oxidative stress reducers, cytoskeletal stabilizers, c-Abl tyrosine kinase inhibitors, anti-apoptotic compounds (e.g., flupirtine) antioxidants (e.g., N-(tert-butyl)hydroxylamine).

Enzyme Replacement Therapy

[00146] Further provided herein arc methods of treating or preventing a disease or disorder in a subject, comprising administering to the subject an effective amount of BMP synthase, a catalytically active fragment or variant thereof, or a nucleic acid encoding BMP synthase, a catalytically active fragment or variant thereof to the subject.

[00147] In some embodiments, the subject has or is suspected of having a disease or disorder characterized by lysosomal dysfunction, decreased lipid catabolism, and/or increased cholesterol accumulation.

[00148] In some embodiments, the subject has or is suspected of having a neurodegenerative disease or disorder or a lysosomal storage disorder, as described above. In some embodiments, the subject has or is suspected of having drug-induced phospholipidosis, familial hypercholesterolemia (FH), a cardiovascular disease, atherosclerosis, obesity, a fatty liver disease, or any combination thereof, as described above.

[00149] In some embodiments, the subject has or is suspected of having a neurodegenerative disease selected from Niemann-Pick disease, frontotemporal dementia (FTD), Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), and Dementia with Lewy Bodies (DLB).

[00150] In some embodiments, the subject has or is suspected of having a neuronal ceroid lipofuscinosis is selected from neuronal ceroid lipofuscinosis type 1 (CLN1), neuronal ceroid lipofuscinosis type 2 (CLN2), neuronal ceroid lipofuscinosis type 3 (CLN3), neuronal ceroid lipofuscinosis type 4 (CLN4), neuronal ceroid lipofuscinosis type 5 (CLN5), neuronal ceroid lipofuscinosis type 6 (CLN6), neuronal ceroid lipofuscinosis type 7 (CLN7), neuronal ceroid lipofuscinosis type 8 (CLN8), neuronal ceroid lipofuscinosis type 9 (CLN9), neuronal ceroid lipofuscinosis type 10 (CLN10), neuronal ceroid lipofuscinosis type 11 (CLN1 1), neuronal ceroid lipofuscinosis type 12 (CLN12), neuronal ceroid lipofuscinosis type 13 (CLN13), and neuronal ceroid lipofuscinosis type 14 (CLN14).

[00151] As described above, BMP synthase, as used herein, refers to any enzyme or fragment thereof which catalyzes a base-exchange reaction between two lysophosphatidylglycerol (LPG) molecules. In some embodiments, the BMP synthase comprises an amino acid sequence of SEQ ID NO: 1. In some embodiments, the BMP synthase comprises an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%) identity to SEQ ID NO: 1. In some embodiments, the BMP synthase comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 1.

[00152] In some embodiments, the BMP synthase, the catalytically active fragment or variant thereof is linked to a polypeptide or molecule configured to bind to a blood-brain barrier (BBB) receptor (e.g., a receptor associated protein (RAP)). For example, the BMP synthase may be linked to molecules such as endogenous ligands or monoclonal antibodies that act to bind exofacial epitopes on BBB receptor-mediated transport systems, triggering internalization of the receptor and of the BMP synthase. In some embodiments, the BMP synthase is linked to a polypeptide comprising a receptor-binding domain from an apolipoprotein, or an engineered variant thereof. In some embodiments, the BMP synthase is linked to a polypeptide comprising a transferrin-receptor binding site.

[00153] In some embodiments, the BMP synthase is linked to a polypeptide comprising an Fc region. In some embodiments, the methods further comprise administering a polypeptide comprising a second Fc region configured to form a dimer with the Fc region. In some embodiments, the polypeptide comprising a first Fc region and/or the polypeptide comprising a second Fc region binds to a blood-brain barrier (BBB) receptor, e.g., endothelial cell receptors resulting in endocytosis of the receptor and bound ligands. In some embodiments, the polypeptide comprising a first Fc region and/or the polypeptide comprising a second Fc region binds to a transferrin receptor.

[00154] The BMP synthase may further comprise a lysosomal targeting moiety, one or more lipid moieties, an epitope tag, one or more (e.g., two, three, four, or more) protein transduction domains or PTDs, or any combination thereof. [00155] In some embodiments, the methods may further comprise administration with one or more additional therapies to treat the disease or disorder, or one or more symptoms of the disease or disorder. The additional therapy may include administration of an additional therapeutic agent or a therapy not connected to administration of another agent including surgery and physical, occupational, and/or speech therapy.

[00156] The additional therapy may be administered at the same time as the initial therapy. For example, either in the same composition or in a separate composition administered at substantially the same time as the first composition. In some embodiments, the additional therapy may precede or follow the treatment of the initial therapy by time intervals ranging from hours to months.

[00157] The methods may further comprise activating autophagy or autophagy related pathways, activating the function or enhancing the levels of transcription factor EB (TFEB), or a combination thereof, as described above. The methods may further comprise treatment with immune modulators and/or neuroprotective compounds, as described above.

BMPS Inhibition

[00158] Also provided herein are methods of treating or preventing a disease or disorder in a subject comprising administering to the subject a therapeutically or prophylactically effective amount of a BMP synthase inhibitor or a composition thereof to the subject. In some embodiments, the disease or disorder is dependent on lipid fusogenic processes and/or enhanced cell fitness. In some embodiments, the disease or disorder is mediated by lysosomal function in excess of normal, non-pathological cellular processes.

[00159] In some embodiments, the disease or disorder is a proliferative disease or disorder, e.g., a disease or disorder that occurs due to abnormal growth or extension by the multiplication or replication of cells. Proliferative diseases or disorders may include benign, premalignant, and malignant cell proliferation. In some embodiments, the proliferative disease is susceptible to cell death involving lysosome membrane permeabilization.

[00160] In some embodiments, the proliferative disease is cancer. The term cancer” refers to a class of diseases characterized by the development of abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy normal body tissues. See, e.g., Stedman’s Medical Dictionary, 25th ed.; Hensyl ed.; Williams & Wilkins: Philadelphia, 1990. [00161] In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer is metastatic cancer. In some embodiments, the disclosed methods result in suppression of elimination of metastasis. In some embodiments, the disclosed methods result in decreased tumor growth. In some embodiments, the disclosed methods prevent tumor recurrence. The cancer may be a primary or secondary cancer in that it can be located where it originated or originate from cancer in other organs (e.g., metastatic cancers), respectively.

[00162] The disclosed methods may be useful to treat a wide variety of cancers including carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma. Exemplary cancers include, but are not limited to, adrenocortical carcinoma, anal cancer, appendix cancer, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, osteosarcoma or malignant fibrous histiocytoma, brain cancer (e.g., brain stem glioma, astrocytoma (e.g., cerebellar, cerebral, etc.), atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, malignant glioma, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma, pineal parenchymal tumors of intermediate differentiation, supratentorial primitive neuroectodermal tumors and/or pineoblastoma, visual pathway and/or hypothalamic glioma, brain and spinal cord tumors, etc.), breast cancer, bronchial tumors, carcinoid tumor (e.g., gastrointestinal, etc.), carcinoma of unknown primary, cervical cancer, chordoma, chronic myeloproliferative disorders, colon cancer, colorectal cancer, embryonal tumors, cancers of the central nervous system, endometrial cancer, ependymoma, esophageal cancer, Ewing family of tumors, eye cancer (e.g., intraocular melanoma, retinoblastoma, etc.), gallbladder cancer, gastric cancer, gastrointestinal tumor (e.g., carcinoid tumor, stromal tumor (gist), stromal cell tumor, etc.), germ cell tumor (e.g., extracranial, extragonadal, ovarian, etc.), gestational trophoblastic tumor, head and neck cancer, hepatocellular cancer, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, islet cell tumors, Kaposi sarcoma, kidney cancer, large cell tumors, laryngeal cancer (e.g., acute lymphoblastic, acute myeloid, etc.), leukemia (e.g., myeloid, acute myeloid, acute lymphoblastic, chronic lymphocytic, chronic myelogenous, multiple myelogenous, hairy cell, etc.), lip and/or oral cavity cancer, liver cancer, lung cancer (e.g., nonsmall cell, small cell, etc.), lymphoma (e.g., AIDS-related, Burkitt, cutaneous Tcell, Hodgkin, non-Hodgkin, primary central nervous system, cutaneous T-cell, Waldenstrom macroglobulinemia, etc.), malignant fibrous histiocytoma of bone and/or osteosarcoma, medulloblastoma, medulloepithelioma, merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases (e.g., myeloproliferative disorders, chronic, etc.), nasal cavity and/or paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cancer; oral cavity cancer, oropharyngeal cancer; osteosarcoma and/or malignant fibrous histiocytoma of bone: ovarian cancer (e.g., ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, etc.), pancreatic cancer (e.g., islet cell tumors, etc.), papillomatosis, paranasal sinus and/or nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pineal parenchymal tumors of intermediate differentiation, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, plasma cell neoplasm/multiple myeloma, pleuropulmonary blastema, prostate cancer, rectal cancer, renal cell cancer, transitional ceil cancer, respiratory tract carcinoma involving the nut gene on chromosome 15, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (e.g., Ewing family of tumors, Kaposi, soft tissue, uterine, etc.), Sezary syndrome, skin cancer (e.g., non-melanoma, melanoma, merkel cell, etc.), small intestine cancer, squamous cell carcinoma, squamous neck cancer with occult primary, metastatic, stomach cancer, supratentorial primitive neuroectodermal tumors, testicular cancer, throat cancer, thymoma and/or thymic carcinoma, thyroid cancer, transitional cell cancer of the renal, pelvis and/or ureter (e.g., trophoblastic tumor, unknown primary site carcinoma, urethral cancer, uterine cancer, endometrial, uterine sarcoma, etc.), vaginal cancer, visual pathway and/or hypothalamic glioma, vulvar cancer, Wilms tumor, and the like. In some embodiments, the cancer is any cancer vulnerable to membrane permeabilization or lysosome related tumorigenicity. In select embodiments, the cancer is lung cancer (e.g., non-small cell, small cell, etc.). In select embodiments, the cancer is pancreatic cancer (e.g., pancreatic adenocarcinoma). In select embodiments, the cancer is prostate cancer. In select embodiments, the cancer is breast cancer (e.g., mammary adenocarcinoma). In select embodiments, the cancer is related to low levels of HSP70.

[00163] In select embodiments, the cancer is a glioma (e.g., astrocytomas, oligodendrogliomas, ependymomas, oligoastrocytoma or another mixed glioma). In some embodiments, the glioma is a glioblastoma. In select embodiments, the cancer is selected from the group consisting of a primary brain tumor, glioblastoma, glioma, meningioma, neurinoma, pituitary adenoma, medulloblastoma, craniopharyngioma, hemangioma, epidermoid, sarcoma and intracranial metastasis from other tumor sources

[00164] In some embodiments, the cancer is a Von Hippel-Lindau (VHL) related cancer (e.g., a VHL syndrome). VHL-related tumors include hemangioblastomas, which are blood vessel tumors of the brain, spinal cord, and retina. The retinal tumors are also called retinal angiomas, which can lead to blindness if not treated in a timely manner. VHL syndrome is also associated with an increased risk of developing clear cell renal cell carcinoma (ccRCC), a specific type of kidney cancer, as well as pancreatic neuroendocrine tumor (pNET). Tumors of the adrenal gland or pheochromocytoma can also develop, with a small number becoming metastatic, meaning they spread to other parts of the body.

[00165] In some embodiments, the cancer is a Kirsten rat sarcoma viral oncogene homologue (K-Ras) driven cancer. A cancer driven by K-Ras can be determined by genetic sequencing of a tumor or cancerous tissue and identifying K-Ras mutations. K-Ras is the most common oncogene with the highest mutation rate among all cancers and is associated with a series of highly fatal cancers, including pancreatic (e.g., pancreatic ductal adenocarcinoma (PDAC)), lung (e.g., non-small cell lung cancer (NSCLC), lung adenocarcinomas), and colorectal (CRC) cancers.

[00166] Examples of noncancerous cellular proliferative disorders include, but are not limited to, fibroadenoma, adenoma, intraductal papilloma, nipple adenoma, adenosis, fibrocystic disease or changes of breast, plasma cell proliferative disorder (PCPD), restenosis, atherosclerosis, rheumatoid arthritis, myofibromatosis, fibrous hamartoma, granular lymphocyte proliferative disorders, benign hyperplasia of prostate, heavy chain diseases (HCDs), lymphoproliferative disorders, psoriasis, idiopathic pulmonary fibrosis, scleroderma, cirrhosis of the liver, TgA nephropathy, mesangial proliferative glomerulonephritis, membranoproliferative glomerulonephritis, hemangiomas, vascular and non-vascular intraocular proliferative disorders, and the like.

[00167] In some embodiments, the disease or disorder is an infectious disease or disorder. For example, a disease or disorder in which an infectious agent (e.g., bacteria, virus, etc.) utilizes lipids for entering a cell. Infectious diseases that can be treated or prevented by the methods of the present invention are caused by infectious agents including, but not limited to, viruses, bacteria, fungi, protozoa, helminths, and parasites. The invention is not limited to treating or preventing infectious diseases caused by intracellular or extracellular pathogens. The infectious disease may be derived from: bacteria, such as Mycobacterium tuberculosis, Chlamydia, Francisella tularensis; DNA viruses, such as Herpesviridae (herpes simplex virus-1, Kaposi's sarcoma-associated virus and Epstein-Barr virus), Papillomaviridae (human papilloma vims), Adenovirus and Hepadnaviridae (Hepatitis B vims), or RNA viruses, such as Retroviridae (human immunodeficiency virus) Flaviviridae (Dengue vims, Hepatitis C virus), Orthomyxoviridae (influenza), and Coronaviridae (human coronavirus and SARS coronavirus). [00168] BMP synthase inhibitors may block access of a substrate or ligand to BMP synthase, disrupt the expression of BMP synthase, block activity of the BMP by binding an allosteric site, or degrade or destabilize BMP synthase. Suitable BMP synthase inhibitors include, but are not limited to, gene silencing oligonucleotides (e.g., an siRNA, an antisense oligonucleotide, dominant-negative, a short-hairpin RNA, a miRNA, a dicer-substrate RNA, a DNAzyme, an guide RNA, or an aptamer targeting the CLN5 gene or the CLN5 messenger RNA), protein configured to bind BMP synthase or a substrate thereof (e.g., an anti-BMP synthase antibody (e.g., a monoclonal, polyclonal, murine, chimeric, humanized, or human antibody targeting an BMP synthase epitope or BMP synthase ligand, thus interfering with BMP synthase activity or ligand binding)), a small molecule inhibitor of BMP synthase, a negative allosteric modulating agent, a protease (e.g., cysteine protease), or combinations thereof.

[00169] In some embodiments, the inhibitor is a dominant negative BMP synthase or variant or fragment thereof. In general, the term “dominant negative” as used herein refers to a protein variant capable of blocking the function of the normal, wild-type protein within the same cell. For example, in some instances, dominant negative activity may occur if the protein variant is capable of binding, or otherwise interacting, with the same cellular components as the wild-type protein, but blocking one or more aspects of the function of the wild-type protein. In particular, the term “dominant negative” as used herein refers to a protein that has been modified so that it interacts with the normal binding partners for that protein, but is lacking the activity (e.g., enzymatic activity) that would normally be present when it forms such interactions.

[00170] In various implementations described herein, the dominant negative activity is due to the modification of or deletion of sequences from the wild-type protein to provide the dominant negative protein. For example, the dominant negative BMP synthase may comprise one or more mutations or substitutions of amino acids in the LPG binding pocket and/or the catalytic active site. Tn specific embodiments, the dominant negative BMP synthase or variant or fragment thereof comprises one or more mutations in cysteine 231, histidine 117, & glutamate 134 in reference to SEQ ID NO: 1.

[00171] In some embodiments the inhibitor is a glycerophosphodiester, also known as glycerophosphoryl diester. Glycerophosphodiester refers to a compound having the general formula , where R is alkyl, alkenyl, alkynyl, alkoxy, acyl, amino, amido, amidino, aryl, azido, carbamoyl, carboxyl, carboxyl ester, cyano, cycloalkyl, cycloalkenyl, guanidino, halo, haloalkyl, haloalkoxy, heteroalkyl, heteroaryl, heterocyclyl, hydroxy, hydrazino, imino, oxo, nitro, phosphate, phosphonate, sulfonic acid, sulfonamido, thiol, thione, thioxo, or combinations thereof. In select embodiments, the glycerophosphodiester is selected from the group consisting of glycerophosphatidyl serine (GPS), glyccrophosphatidylcthanolaminc (GPE), glycosylphosphatidylinositol (GPI), glycerophosphoglycerol (GPG), glycerophosphatidylcholine (GPC), and combinations thereof. [00172] In some embodiments, the inhibitor comprises a negative allosteric modulating agent. The negative allosteric modulating agent may bind to any location outside of the substrate binding pocket (e.g., pocket including cysteine 231, histidine 117, & glutamate 134). In some embodiments, the negative allosteric modulating agent is configured to bind to a binding site of BMP synthase, a catalytically active fragment or variant thereof, comprising one, two or all residues at positions 73, 143, and 296 in reference to SEQ ID NO: 1. In some embodiments, the negative allosteric modulating agent is configured to bind to a binding site of BMP synthase, a catalytically active fragment or variant thereof, comprising one, two or all of K73, N143, and K296, in reference to SEQ ID NO: 1.

[00173] As such, also disclosed herein are compounds which are configured to bind to a binding site of BMP synthase, a catalytically active fragment or variant thereof, comprising one, two or all residues at positions 73, 143, and 296, in reference to SEQ ID NO: 1. In some embodiments, the compound is configured to bind to a binding site of BMP synthase comprising one, two or all of K73, N143, and K296, in reference to SEQ ID NO: 1. [00174] Also disclosed are methods of inhibiting BMP synthesis comprising binding a compound to the binding site of BMP synthase, a catalytically active fragment or variant thereof, comprising one, two or all residues at positions 73, 143, and 296, in reference to SEQ ID NO: 1. Also disclosed are methods of inhibiting BMP synthesis comprising binding a compound to a binding site of BMP synthase, a catalytically active fragment or variant thereof, comprising residues at positions one, two or all ofK73, N143, and K296, in reference to SEQ ID NO: 1. [00175] In some embodiments, the methods may further comprise administration with one or more additional therapies to treat the disease or disorder, or one or more symptoms of the disease or disorder. The additional therapy may include administration of an additional therapeutic agent or a therapy not connected to administration of another agent including surgery, immunotherapy, radiotherapy.

[00176] The additional therapy may be administered at the same time as the initial therapy. For example, either in the same composition or in a separate composition administered at substantially the same time as the first composition. In some embodiments, the additional therapy may precede or follow the treatment of the initial therapy by time intervals ranging from hours to months.

[00177] In some embodiments, the additional therapeutic agent comprises an immune modulator, a chemotherapeutic agent, a steroid, an analgesic, an antimicrobial agent, an immunotherapy, or a combination thereof.

[00178] Exemplary immune modulators include: indoleamine 2,3-dioxygenase (IDO) inhibitors and analogs thereof, such as, epacadostat, BMS-986205, indoximod, PF-06840003, and analogs thereof; signal transducer and activator of transcription 3 (Stat3) inhibitors and analogs thereof, such as, SM-36 and its analogs; toll-like receptor (TLR) agonists and analogs thereof, such as, imiquimod, resiquimod, selgantolimod, gardiquimod, SM-360320, TMX-101, TMX-202, TMX-302, TMX-306, GSK2245035, CL097, 852A, AZD-8848, DSP-3025, GS- 9620, R07020531, RO6871765, ANA773, DSP-0509, NJH395, BNT411, TQ-A3334, JNJ-4964, LHC165, CV8102, VTX-1463, VTX-2337, IMO-8400, IMO-3100, IRS-954, and analogs thereof; and statins or other lipid-lowering medications and analogs thereof, such as, atorvastatin, pravastatin, fluvastatin, simvastatin, lovastatin, mevastatin, pitavastatin, rosuvastatin, and analogs thereof. [00179] In some embodiments, the additional therapeutic agent comprises at least one chemotherapeutic agent. As used herein, the term “chemotherapeutic” or “anti-canccr drug” includes any small molecule or other drug used in cancer treatment or prevention. Chemotherapeutic s include, but are not limited to, cyclophosphamide, methotrexate, 5- fluorouracil, doxorubicin, docetaxel, daunorubicin, bleomycin, vinblastine, dacarbazine, cisplatin, paclitaxel, raloxifene hydrochloride, tamoxifen citrate, abemacicilib, afinitor (Everolimus), alpelisib, anastrozole, pamidronate, anastrozole, exemestane, capecitabine, epirubicin hydrochloride, eribulin mesylate, toremifene, fulvestrant, letrozole, gemcitabine, goserelin, ixabepilone, emtansine, lapatinib, olaparib, megestrol, neratinib, palbociclib, ribociclib, talazoparib, thiotepa, toremifene, methotrexate, and tucatinib. In select embodiments, the chemotherapeutic agent comprises paclitaxel.

[00180] In some embodiments of the methods disclosed herein, the additional therapeutic agent comprises an antimicrobial (e.g., antiviral or antibacterial) agent. In some embodiments, the additional antimicrobial agent is an antiviral agent, including but not limited to, abacavir, acyclovir, adefovir, amantadine, amprenavir, atazanavir, baloxavir marboxil, bictegravir, boceprevir, bulevirtide, cidofovir, cobicistat, daclatasvir, darunavir, delavirdine, didanosine, docosanol, dolutegravir, doravirine, edoxudine, efavirenz, elvitegravir, emtricitabine, enfuvirtide, entecavir, etravirine, famciclovir, fomivirsen, fosamprenavir, foscarnet, ganciclovir, ibacitabine, ibalizumab, idoxuridine, imiquimod, imunovir, indinavir, lamivudine, letermovir, lopinavir, loviride, maraviroc, methisazone, moroxydine, nelfinavir, nevirapine, nexavir, nitazoxanide, oseltamivir, penciclovir, peramivir, penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir, remdesivir, ribavirin, rilpivirine, rilpivirine, rimantadine, rintatolimod, ritonavir, saquinavir, simeprevir, sofosbuvir, stavudine, taribavirin, telaprevir, telbivudine, tenofovir (e.g., tenofovir alafenamide or tenofovir disoproxil), tipranavir, trifluridine, tromantadine, umifenovir, valaciclovir, valganciclovir, vicriviroc, vidarabinc. zalcitabine, zanamivir, and zidovudine, and any combination thereof.

[00181] In some embodiments, the antimicrobial agent is an antibacterial agent. Exemplary antibacterial agents include sulfonamides, amphenicols, spectinomycin, trimethoprim, glycylcyclines, macrolides (e.g., erythromycin, clarithromycin, azithromycin, roxithromycin), oxazolidinones (e.g., linezolid), tetracyclines (e.g., doxycycline, tetracycline, minocycline), - lactams (e.g., penicillin, methicillin, cioxacillin), carbapenems (e.g., imipenem, meropenem, aztreonam), aminoglycosides (e.g., gentamicin, tobramycin, amikacin), quinolones and fluoroquinolones (e.g., levofloxacin, ciprofloxacin, moxifloxacin), glycopeptides (e.g., vancomycin), polymyxins (e.g., polymyxin, colistin).

[00182] In some embodiments, the additional therapy includes immunotherapy. Immunotherapies include chimeric antigen receptor (CAR) T-cell or T-cell transfer therapies, cytokine therapy, immunomodulators, cancer vaccines, or administration of antibodies (e.g., monoclonal antibodies).

[00183] In some embodiments, the immunotherapy comprises administration of antibodies. The antibodies may target antigens either specifically expressed by tumor cells or antigens shared with normal cells. In some embodiments, the immunotherapy may comprise an antibody targeting, for example, CD20, CD33, CD52, CD30, HER (also referred to as erbB or EGFR), VEGF, CTLA-4 (also referred to as CD 152), epithelial cell adhesion molecule (EpCAM, also referred to as CD326), and PD-1/PD-L1. Suitable antibodies include, but are not limited to, rituximab, blinatumomab, trastuzumab, gemtuzumab, alemtuzumab, ibritumomab, tositumomab, bevacizumab, cetuximab, panitumumab, ofatumumab, ipilimumab, brentuximab, pertuzumab, and the like). In some embodiments, the additional therapeutic agent may comprise anti-PD- 1/PD-L1 antibodies, including, but not limited to, pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, and ipilimumab. The antibodies may also be linked to a chemotherapeutic agent. Thus, in some embodiments, the antibody is an antibody-drug conjugate.

[00184] The immunotherapy (e.g., administration of antibodies) may be administered to a subject by a variety of methods. In any of the uses or methods described herein, administration may be by various routes known to those skilled in the art, including without limitation oral, inhalation, intravenous, intramuscular, topical, subcutaneous, systemic, and/or intraperitoneal administration to a subject in need thereof. The immunotherapy may be administered by parenteral administration (including, but not limited to, subcutaneous, intramuscular, intravenous, intraperitoneal, intracardiac and intraarticular injections).

Administration

[00185] In the methods disclosed herein, administration may be by any convenient route of administration, whether systemically/peripherally or at the site of desired action, including but not limited to, oral (e.g., by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., an aerosol, e.g., through mouth or nose); rectal; vaginal; parenteral (e.g., by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal injection); or by implant of a depot, for example, subcutaneously or intramuscularly.

[00186] In some embodiments, the administration is via the cerebrospinal fluid. In select embodiments, the administration comprises intrathecal, intracistemal, intranasal, or intracerebroventricular administration. Intrathecal drug administration can be accomplished by lumbar puncture or by an implanted intrathecal drug delivery device (IDDD).

Intracerebroventricular administration facilitates administration of drugs into a lateral cerebral ventricle, e.g., by direct injection or via an implanted device (reservoir and catheter).

[00187] In some embodiments, the methods disclosed herein may further comprise opening or increasing the permeability of the blood-brain barrier (BBB) prior to administration, particularly for administration methods which target the agent(s) to the bloodstream. For example, mannitol has been used as an osmotic substance, for increasing BBB permeability. Convection enhanced delivery (CED) is another technique that has been explored to bypass the BBB. CED is performed by first inserting a small catheter directly into the targeted brain region and then by slowly infusing the drug directly into the tissue, thereby bypassing the BBB. Pulsed ultrasound (US) or ultrasound beams temporarily disrupts the BBB. Recently administration of gold nanoparticles having the therapeutic agent followed by laser pulses modulated the BBB permeability to successfully deliver a variety of agents to the brain.

[00188] In some embodiments, the BMP synthase, BMP synthase activators, and BMP synthase inhibitors described herein may be delivered using nanoscale drug delivery platforms mainly including lipid- and polymer-based nanoparticles (NPs) that assure a controlled and improved release of their cargo by protecting loaded drugs from being metabolized and result delivery to the brain.

[00189] Any of the BMP synthase, a catalytically active fragment or variant thereof, or a nucleic acid encoding BMP synthase or a catalytically active fragment or variant thereof, BMP synthase activators, and BMP synthase inhibitors or ex vivo treated cells may be administered with a pharmaceutically acceptable carrier or excipient as a pharmaceutical composition. In some embodiments, the BMP synthase, a catalytically active fragment or variant thereof, or a nucleic acid encoding BMP synthase or a catalytically active fragment or variant thereof, BMP synthase activators, and BMP synthase inhibitors or ex vivo treated cells may be mixed with a pharmaceutically acceptable carrier to form pharmaceutical compositions, which are also within the scope of the present disclosure.

[00190] The phrase “pharmaceutically acceptable,” as used in connection with compositions and/or cells of the present disclosure, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a subject (e.g., a mammal, a human). Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans. “Acceptable” means that the carrier is compatible with the active ingredient of the composition (e.g., the nucleic acids, vectors, cells, or therapeutic antibodies) and does not negatively affect the subject to which the composition(s) are administered. Any of the pharmaceutical compositions and/or cells to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formations or aqueous solutions.

[00191] Pharmaceutically acceptable carriers, including buffers, are well known in the art, and may comprise phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; and/or non-ionic surfactants. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.

[00192] When utilized as a method of treatment, the effective amount and/or dosage of the BMP synthase, a catalytically active fragment or variant thereof, or a nucleic acid encoding BMP synthase or a catalytically active fragment or variant thereof, BMP synthase activators, and BMP synthase inhibitors or ex vivo treated cells may depend on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. In some embodiments, the effective amount alleviates, relieves, ameliorates, improves, reduces the symptoms, or delays the progression of any disease or disorder in the subject. In some embodiments, the subject is a human.

[00193] In the context of the present disclosure insofar as it relates to any of the disease conditions recited herein, the terms “treat,” “treatment,” and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition. Within the meaning of the present disclosure, the term “treat” also denotes to arrest, delay the onset (e.g., the period prior to clinical manifestation of a disease) and/or reduce the risk of developing or worsening a disease, [00194] It will be appreciated that appropriate dosages can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present disclosure. The selected dosage level will depend on a variety of factors including, but not limited to, the relative activity of the BMP synthase, catalytically active fragment or variant thereof, or a nucleic acid encoding BMP synthase or a catalytically active fragment or variant thereof, BMP synthase activators, and BMP synthase inhibitors or ex vivo treated cells, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient. The amount and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects.

[00195] Administration in vivo can be in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.

[00196] The BMP synthase, catalytically active fragment or variant thereof, a nucleic acid encoding BMP synthase or a catalytically active fragment or variant thereof, BMP synthase activators, or BMP synthase inhibitors may be administered once, on a continuous basis (e.g. by an intravenous drip), or on a periodic/intermittent basis, including about once per hour, about once per two hours, about once per four hours, about once per eight hours, about once per twelve hours, about once per day, about once per two days, about once per three days, about twice per week, about once per week, and about once per month. The composition may be administered until a desired reduction of symptoms is achieved.

[00197] Other therapies, as included in the above methods, may be used in combination with the BMP synthase, a catalytically active fragment or variant thereof, a nucleic acid encoding BMP synthase or a catalytically active fragment or variant thereof, BMP synthase activators, and BMP synthase inhibitors. Administered “in combination,” as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

[00198] The methods described herein may be applied to cell populations in vivo or ex vivo. “In vivo” means within a living individual, as within an animal or human. In this context, the methods described herein may be used therapeutically in an individual. “Ex vivo” means outside of a living individual. Examples of ex vivo cell populations include in vitro cell cultures and biological samples including fluid or tissue samples obtained from individuals. Such samples may be obtained by methods well known in the art. Exemplary biological fluid samples include blood, cerebrospinal fluid, urine, and saliva. In this context, the methods described herein may be used for a variety of purposes, including therapeutic and experimental purposes. For example, the methods may be used ex vivo to determine the optimal schedule and/or dosing of administration for a given indication, cell type, individual, and other parameters. Information gleaned from such use may be used for experimental purposes or in the clinic to set protocols for in vivo treatment. In some embodiments, the administering comprises transplantation of ex vivo treated cells.

Nucleic acids

[00199] The present disclosure also provides for DNA segments encoding the proteins (e.g., BMP synthase or active fragment or variant thereof or dominant negative variant thereof, progranulin, Fc polypeptides, etc.) and nucleic acids (e.g., nucleic acids and vectors encoding the disclosed proteins, gene silencing oligonucleotides) disclosed herein, vectors containing these segments and cells containing the vectors. The vectors may be used to propagate the segment in an appropriate cell and/or to allow expression from the segment (e.g., an expression vector). The person of ordinary skill in the art would be aware of the various vectors available for propagation and expression of a nucleic acid sequence.

[00200] The nucleic acid encoding the proteins (e.g., BMP synthase or active fragment or variant thereof or dominant negative variant thereof, progranulin, Fc polypeptides, etc.) disclosed herein may be any nucleic acid including DNA, RNA, or combinations thereof. In some embodiments, the nucleic acid encoding the proteins (e.g., BMP synthase or active fragment or variant thereof or dominant negative variant thereof, progranulin, Fc polypeptides, etc.) comprises a messenger RNA or a vector.

[00201] In certain embodiments, engineering the nucleic acid for use in eukaryotic cells may involve codon-optimization. It will be appreciated that changing native codons to those most frequently used in mammals allows for maximum expression of the system proteins in mammalian cells (e.g., human cells). Such modified nucleic acid sequences are commonly described in the art as “codon-optimized,” or as utilizing “mammalian-preferred” or “humanpreferred” codons. In some embodiments, the nucleic acid sequence is considered codon- optimized if at least about 60% (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%) of the codons encoded therein are mammalian preferred codons. [00202] The present disclosure further provides engineered, non-naturally occurring vectors and vector systems, which can encode the proteins (c.g., BMP synthase or active fragment or variant thereof or dominant negative variant thereof, progranulin, Fc polypeptides, etc.) and nucleic acids (e.g., nucleic acids and vectors encoding the disclosed proteins, gene silencing oligonucleotides) disclosed herein. The vector(s) can be introduced into a cell that is capable of expressing the polypeptide encoded thereby, including any suitable prokaryotic or eukaryotic cell.

[00203] The vectors of the present disclosure may be delivered to a eukaryotic cell in a subject. Modification of the eukaryotic cells can take place in a cell culture, where the method comprises isolating the eukaryotic cell from a subject prior to the modification. In some embodiments, the method further comprises returning said eukaryotic cell and/or cells derived therefrom to the subject.

[00204] Viral and non- viral based gene transfer methods can be used to introduce nucleic acids encoding the proteins (e.g., BMP synthase or active fragment or variant thereof or dominant negative variant thereof, progranulin, Fc polypeptides, etc.) and nucleic acids (e.g., nucleic acids and vectors encoding the disclosed proteins, gene silencing oligonucleotides) disclosed herein into cells, tissues, or a subject. Such methods can be used to administer nucleic acids encoding the proteins (e.g., BMP synthase or active fragment or variant thereof or dominant negative variant thereof, progranulin, Fc polypeptides, etc.) and nucleic acids (e.g., nucleic acids and vectors encoding the disclosed proteins, gene silencing oligonucleotides) disclosed herein to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, cosmids, RNA (e.g., a transcript of a vector described herein), a nucleic acid, and a nucleic acid complexed with a delivery vehicle. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. Viral vectors include, for example, retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors.

[00205] In certain embodiments, plasmids that are non-replicative, or plasmids that can be cured by high temperature may be used, such that the nucleic acid encoding the proteins (e.g., BMP synthase or active fragment or variant thereof or dominant negative variant thereof, progranulin, Fc polypeptides, etc.) and nucleic acids (e.g., nucleic acids and vectors encoding the disclosed proteins, gene silencing oligonucleotides) disclosed herein may be removed from the cells under certain conditions.

[00206] A variety of viral constructs may be used to deliver the proteins (e.g., BMP synthase or active fragment or variant thereof or dominant negative variant thereof, progranulin, Fc polypeptides, etc.) and nucleic acids (e.g., nucleic acids and vectors encoding the disclosed proteins, gene silencing oligonucleotides) disclosed herein to the targeted cells and/or a subject. Nonlimiting examples of such recombinant viruses include recombinant adeno-associated virus (AAV), recombinant adenoviruses, recombinant lentiviruses, recombinant retroviruses, recombinant herpes simplex viruses, recombinant poxviruses, phages, etc. The present disclosure provides vectors capable of integration in the host genome, such as retrovirus or lentivirus. See, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989; Kay, M. A., et al., 2001 Nat. Medic. 7(1 ):33-40; and Walther W. and Stein U„ 2000 Drugs, 60(2): 249-71, incorporated herein by reference.

[00207] In one embodiment, a DNA segment encoding the proteins (e.g., BMP synthase or active fragment or variant thereof or dominant negative variant thereof, progranulin, Fc polypeptides, etc.) and nucleic acids (e.g., nucleic acids and vectors encoding the disclosed proteins, gene silencing oligonucleotides) disclosed herein is contained in a plasmid vector that allows expression of the protein(s) and subsequent isolation and purification of the protein produced by the recombinant vector. Accordingly, the proteins can be purified following expression, obtained by chemical synthesis, or obtained by recombinant methods.

[00208] In certain embodiments, vectors of the present disclosure can drive the expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, Nature (1987) 329:840, incorporated herein by reference) and pMT2PC (Kaufman, et al., EMBO J. (1987) 6:187, incorporated herein by reference). When used in mammalian cells, the expression vector's control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd eds., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference. [00209] Vectors of the present disclosure can comprise any of a number of promoters known to the art, wherein the promoter is constitutive, rcgulatablc or inducible, cell type specific, tissuespecific, or species specific. In addition to the sequence sufficient to direct transcription, a promoter sequence of the invention can also include sequences of other regulatory elements that are involved in modulating transcription (e.g., enhancers, Kozak sequences and introns). Many promoter/regulatory sequences useful for driving constitutive expression of a gene are available in the art and include, but are not limited to, for example, CMV (cytomegalovirus promoter), EFla (human elongation factor 1 alpha promoter), SV40 (simian vacuolating virus 40 promoter), PGK (mammalian phosphoglycerate kinase promoter), Ubc (human ubiquitin C promoter), human beta-actin promoter, rodent beta-actin promoter, CBh (chicken beta-actin promoter), CAG (hybrid promoter contains CMV enhancer, chicken beta actin promoter, and rabbit betaglobin splice acceptor), TRE (Tetracycline response element promoter), Hl (human polymerase III RNA promoter), U6 (human U6 small nuclear promoter), and the like. Additional promoters that can be used for expression of the components of the present system, include, without limitation, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Maloney murine leukemia virus (MMLV) LTR, myeoloproliferative sarcoma virus (MPSV) LTR, spleen focus-forming virus (SFFV) LTR, the simian virus 40 (SV40) early promoter, herpes simplex tk virus promoter, elongation factor 1- alpha (EFl-a) promoter with or without the EFl-a intron. Additional promoters include any constitutively active promoter. Alternatively, any regulatable promoter may be used, such that its expression can be modulated within a cell.

[00210] Moreover, inducible and tissue specific expression of an RNA or protein can be accomplished by placing the nucleic acid encoding such a molecule under the control of an inducible or tissue specific promoter/regulatory sequence. Examples of tissue specific or inducible promoter/regulatory sequences which are useful for this purpose include, but are not limited to, the rhodopsin promoter, the MMTV LTR inducible promoter, the SV40 late enhancer/promoter, synapsin 1 promoter, ET hepatocyte promoter, GS glutamine synthase promoter and many others. Various ubiquitous as well as tissue-specific promoters and tumorspecific are commercially available, for example from InvivoGen. In addition, promoters which are well known in the art can be induced in response to inducing agents such as metals, glucocorticoids, tetracycline, hormones, and the like, are also contemplated for use with the invention. Thus, it will be appreciated that the present disclosure includes the use of any promotcr/rcgulatory sequence known in the art that is capable of driving expression of the desired protein or RNA operably linked thereto.

[00211] The vectors of the present disclosure may direct expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Such regulatory elements include promoters that may be tissue specific or cell specific. The term “tissue specific” as it applies to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g., seeds) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue. The term “cell type specific” as applied to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest in a specific type of cell in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue. The term “cell type specific” when applied to a promoter also means a promoter capable of promoting selective expression of a nucleotide sequence of interest in a region within a single tissue. Cell type specificity of a promoter may be assessed using methods well known in the art, e.g., immunohistochemical staining.

[00212] Additionally, the vector may contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in host cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; 5’-and 3 ’-untranslated regions for mRNA stability and translation efficiency from highly-expressed genes like a-globin or P-globin; SV40 polyoma origins of replication and ColEl for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA; a “suicide switch” or “suicide gene” which when triggered causes cells carrying the vector to die (e.g., HSV thymidine kinase, an inducible caspase such as iCasp9), and reporter gene for assessing expression of the chimeric receptor. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art. Selectable markers also include chloramphenicol resistance, tetracycline resistance, spectinomycin resistance, streptomycin resistance, erythromycin resistance, rifampicin resistance, bleomycin resistance, thermally adapted kanamycin resistance, gentamycin resistance, hygromycin resistance, trimethoprim resistance, dihydrofolate reductase (DHFR), GPT; the URA3, HIS4, LEU2, and TRP1 genes of S. ccrcvisiac.

[00213] When introduced into the cell, the vectors may be maintained as an autonomously replicating sequence or extrachromosomal element or may be integrated into host DNA.

[00214] The present proteins (e.g., BMP synthase or active fragment or variant thereof or dominant negative variant thereof, progranulin, Fc polypeptides, etc.), nucleic acids encoding these proteins, and compositions comprising the proteins and/or nucleic acids described herein may be delivered by any suitable means. In certain embodiments, they are delivered in vivo, as described above. In other embodiments, they are delivered to isolated/cultured cells (e.g., autologous iPS cells) in vitro (e.g., to provide modified cells useful for in vivo delivery to patients afflicted with a disease or condition).

[00215] As described above, the proteins (e.g., BMP synthase or active fragment or variant thereof or dominant negative variant thereof, progranulin, Fc polypeptides, etc.) or nucleic acids encoding thereof may be introduced into cells by methods known in the art. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a mammalian cell (e.g., a cell of a non-human primate or a human cell). Accordingly, provided herein are cells comprising the disclosed proteins (e.g., a BMP synthase, a catalytically active fragment or variant thereof or a dominant negative variant thereof) or nucleic acids encoding thereof.

[00216] Vectors according to the present disclosure can be transformed, transfected, or otherwise introduced into a wide variety of host cells. Transfection refers to the taking up of a vector by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, lipofectamine, calcium phosphate co-precipitation, electroporation, DEAE-dextran treatment, microinjection, viral infection, and other methods known in the art. Transduction refers to entry of a virus into the cell and expression (e.g., transcription and/or translation) of sequences delivered by the viral vector genome. In the case of a recombinant vector, “transduction” generally refers to entry of the recombinant viral vector into the cell and expression of a nucleic acid of interest delivered by the vector genome.

[00217] Any of the vectors comprising a nucleic acid sequence that encodes proteins (e.g., BMP synthase or an active fragment or variant thereof, progranulin, Fc polypeptides, etc.) and nucleic acids (e.g., nucleic acids and vectors encoding the disclosed proteins, gene silencing oligonucleotides) disclosed herein is also within the scope of the present disclosure. Such a vector may be delivered into host cells by a suitable method. Methods of delivering vectors to cells are well known in the art and may include DNA or RNA electroporation, transfection reagents such as liposomes or nanoparticles to delivery DNA or RNA; delivery of DNA, RNA, or protein by mechanical deformation (see, e.g., Sharei et al. Proc. Natl. Acad. Sci. USA (2013) 110(6): 2082-2087, incorporated herein by reference); or viral transduction. In some embodiments, the vectors are delivered to host cells by viral transduction. Nucleic acids can be delivered as pail of a larger construct, such as a plasmid or viral vector, or directly, e.g., by electroporation, lipid vesicles, viral transporters, microinjection, and biolistics (high-speed particle bombardment). Similarly, the vector can be delivered by any method appropriate for introducing nucleic acids into a cell.

[00218] Additionally, delivery vehicles such as nanoparticle- and lipid-based mRNA or protein delivery systems can be used. Further examples of delivery vehicles include lentiviral vectors, ribonucleoprotein (RNP) complexes, lipid-based delivery system, gene gun, hydrodynamic, electroporation or nucleofection microinjection, and biolistics. Various gene delivery methods are discussed in detail by Nayerossadat et al. (Adv Biomed Res. 2012; 1: 27) and Ibraheem et al. (Int J Pharm. 2014 Jan 1 ;459( l-2):70-83), incorporated herein by reference.

Kits

[00219] In another aspect, the disclosure provides kits or systems comprising a BMP synthase or an active fragment or variant thereof, a nucleic acid encoding the BMP synthase or an active fragment or variant thereof, or a composition thereof, and instructions for using the protein, nucleic acid, or composition. The kits or systems can also comprise other agents and/or products co-packaged, co-formulated, and/or co-delivered with other components. For example, the kits or systems may further comprise a BMP synthase activator (e.g., HSP70 or cofactors thereof) or nucleic acid encoding thereof, as appropriate, and/or one or more additional therapies to treat a neurodegenerative disease or disorder, a lysosomal storage disorder, drug-induced phospholipidosis, familial hypercholesterolemia (FH), a cardiovascular disease, atherosclerosis, obesity, a fatty liver disease, or any combination thereof. For example, a drug manufacturer, a drug reseller, a physician, a compounding shop, or a pharmacist can provide a kit comprising another agent (e.g., immune modulators and/or neuroprotective compounds) for delivery to a patient. Individual member components of the kits may be physically packaged together or separately.

[00220] In another aspect, the disclosure provides kits or systems comprising a BMP synthase inhibitor. In some embodiments, the inhibitor comprises a protein configured to bind BMP synthase or a substrate thereof, a gene silencing oligonucleotide, a small molecule inhibitor of BMP synthase, a negative allosteric modulating agent, a protease, a dominant negative BMPS or variant or fragment thereof, or a combination thereof, or nucleic acid encoding thereof, as appropriate. The kit may further comprise one or more additional therapies to treat cancer and/or an infectious disease. For example, a drug manufacturer, a drug reseller, a physician, a compounding shop, or a pharmacist can provide a kit comprising another agent (e.g., a chemotherapeutic, a monoclonal antibody, a pain reliever, a steroid, an anti-emetic, an antimicrobial agent) for delivery to a patient.

[00221] Individual member components of the kits may be physically packaged together or separately.

[00222] The kits can also comprise instructions for using the components of the kit. The instructions are relevant materials or methodologies pertaining to the kit. The materials may include any combination of the following: background information, list of components, brief or detailed protocols for using the compositions, trouble-shooting, references, technical support, and any other related documents. Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.

[00223] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Individual member components of the kits may be physically packaged together or separately.

[00224] It is understood that the disclosed kits or systems can be employed in connection with the disclosed methods. The kits or systems may further contain containers or devices for use with the methods or compositions disclosed herein, for example delivery devices (e.g., syringes and the like).

[00225] The following examples further illustrate aspects of the disclosure, but should not be construed as in any way limiting its scope. EXAMPLES

Materials and Methods

[00226] L-a-Glycerophosphorylcholine (GPC), amiodarone hydrochloride, D-(+)-glucose, and valproic acid sodium salt were purchased from Sigma; (18:1/18:1) BMP (S,S), (18:1/18:1) BMP (S,R), 14:0 Lyso PG, 16:0 Lyso PG, 18:0 Lyso PG, 18:1 Lyso PG, 18:1 Lyso PC, 18:1 Lyso PE, 18:1 Lyso PI, 18:1 Lyso PS, (16:0/18:1) PC (POPC), and (16:0/16:0) PG were purchased from Avanti; Glycerophosphoinositol (GPI) was purchased from Echelon Biosciences; Filipin III was purchased from Cayman Chemical; IRDye 800CW anti-mouse and anti-rabbit secondary antibodies were purchased from LICOR; Inactivated fetal bovine serum, DMEM high glucose, Expi293 expression media, Alexa Fluor 488 NHS Ester, LysoTracker™ RED DND-99, Hoechst 33342, Freestyle 293 expression media, and HisPur Ni-NTA resin from Thermo Fisher Scientific; FectoPRO Transfection Kit from Polyplus; XtremeGene9 DNA Transfection Reagent from Roche; and Protein label Kit RED-NHS, Monolith NT.l 15 premium capillaries, and Tycho NT.6 capillaries from Nanotemper Technologies, Inc. Antibody sources are shown in the table below.

[00227] HEK293T cells were acquired from ATCC. Expi293F cells were a gift from the Peter Kim lab (Stanford University). Codon optimized CLN5 WT and mutant geneblocks were cloned into the pRK5 vector.

[00228] Cell culture HEK293Ts cells and their derivatives were cultured in DMEM base media with 10% inactivated fetal calf serum (Thermo Fisher Scientific) supplemented with 2 mM glutamine, penicillin, and streptomycin (Thermo Fisher Scientific). Expi293 cells were cultured in 2/3 FreeStyle: 1/3 Expi293 expression media (Thermo Fisher Scientific). HEK293Ts cells and their derivatives were maintained at 37°C and 5% CO2. Expi293 cells were maintained at 37°C and 8% CO2. For all LysoIP experiments using HEK293T cell lines, cells were cultured in DMEM base media supplemented with 2 mM glutamine, penicillin, streptomycin, and 4 nM of LysoTracker Red DND-99 for one hour before processing via methods described in Abu- Remaileh et al. (Science 358, 807-813 (2017)). [00229] Human iPSCs and iPSC-derived neurons. Male WTC11 human induced pluripotent stem cells (iPSCs) expressing mNGN2 under a doxycyclinc-induciblc system in the AAVS1 safe harbor locus were used in compliance with Stem Cell Research Oversight (SCRO) ethical standards.

[00230] The iPSCs were cultured in StemFlex™ Medium (Cat. No. A3349401) coated with rhLaminin (Cat. No. A29248) that was diluted in 1XDPBS with 2.5 pg final concentration. To initiate differentiation, the iPSCs were enzymatically detached using ReLeSR™ (Cat. No. 100- 0484), and the pelleted cells were resuspended in N2 Pre-Differentiation Medium containing Knockout DMEM/F12 (Gibco/Thermo Fisher Scientific), IX MEM Non-Essential Amino Acids (Sigma), IX N2 Supplement (Gibco/Thermo Fisher Scientific; Cat. No. 17502-048), lOng/mL NT-3 (PeproTech; Cat. No. 450-03), lOng/mL BDNF (PeproTech; Cat. No. 450-02), 1 pg/mL Mouse Laminin (Thermo Fisher Scientific; Cat. No. 23017-015), lOnM ROCK inhibitor, and 2pg/mL doxycycline hydrochloride to induce mNGN2 expression.

[00231] The iPSCs were seeded onto rhLaminin-coated wells of a 6-well plate at a density of 1.5X106cells per well and were cultured in 2mL of N2 Pre-Differentiation Medium. After three days, referred to as Day 0, pre-differentiated cells were released with Accutase, centrifuged, and the pelleted cells were resuspended in Classic Neuronal Medium. The Classic Neuronal Medium contained a 1:1 mixture of half DMEM/F12 (Gibco/Thermo Fisher Scientific; Cat. No. 11320- 033) and half NeurobasaLA (Gibco/Thermo Fisher Scientific; Cat. No. 10888-022) as the base, IX MEM Non-Essential Amino Acids, 0.5X GlutaMAX Supplement (Gibco/Thermo Fisher Scientific; Cat. No. 35050-061), 0.5X N2 Supplement, 0.5X B27 Supplement (Gibco/Thermo Fisher Scientific; Cat. No. 17504-044), lOng/mL NT-3, lOng/mL BDNF, Ipg/mL Mouse Laminin, and 2pg/mL doxycycline hydrochloride.

[00232] The pre-differentiated cells were then counted and seeded at a density of 3.0 x 105cells per well in a Poly-D-Lysine coated 12-well plate containing 2mL of Classic Neuronal Medium, or at a density of 1.5 x 10⁵ cells per Poly-D-Lysine coated 24- well plate with ImL medium. On Day 7, half of the medium was replaced with an equal volume of fresh Classic Neuronal Medium without doxycycline, and the cells were further cultured for 7 more days. On day 14, the differentiated cells were collected for future experiments.

[00233] Virus production and transduction HEK293T cells were transfected with lentiviral plasmids along with packaging plasmids VPR and VSV-G envelope using XtremeGene9 transfection reagent. After 16 hours, the culture medium was replaced with DMEM supplemented with 30% inactivated fetal bovine scrum. After 48 hours, the supernatant was harvested, centrifuged for 5 min at 230 g to remove cells, and frozen at -80°C. Stable expression cell lines were prepared by first plating 1,000,000 WT or CLN5 KO HEK293T cells in 6- well plates in DMEM with 10% inactivated fetal bovine serum, 8 pg/mL polybrene, and 100-250 uL of virus-containing media. Spin infection was then performed at 2,200 RPM for 45 minutes at 37°C. Following a 16-hour incubation, virus-containing medium was replaced with fresh culture medium containing puromycin (Sigma).

[00234] Generation of knock-out cell lines using CRISPR-Cas9 technology CLN5 KO HEK293Ts cells were generated using the Synthego Gene Knockout Kit v2 with the following mixed guide sgRNAs sequences: AAUAAGGAUCAGGUUUUGGA (SEQ ID NO: 7) CAUCAUCACCCUCCAUAACU (SEQ ID NO: 8), and GCAUCCUUACCAAGUGUCCC (SEQ ID NO: 9). Briefly, 20 pmol Cas9 (IDT) and 180 pmol sgRNA were incubated at room temperature for 10 minutes. In the meantime, HEK293Ts were washed with PBS, dissociated using enzyme-free dissociation buffer (ThermoFisher), and counted to determine density. A suspension of 150,000 cells was added to the freshly assembled RNP complexes, transferred to a Lonza Nucleocuvtte^Tm, and electroporated with the CM- 130 program code. Cells were suspended in growth medium and transferred to a 24-well plate. Single cell populations were obtained using limited dilution, and genetic knockouts were confirmed by Synthego ICE CRISPR analysis and western blot.

[00235] CLN5 KO iPSCs were generated using the following sgRNA sequence from Synthego: AAGGGGCAUCCAUUUCAGGU (SEQ ID NO: 10). Briefly, 0.6 pg Cas9 (IDT), 3 pg sgRNA, 16.4 pL P3 Primary Cell NucleofectorTMSolution (Lonza), 3.6 pL Supplement 1 (Lonza), and 300,000 cells were incubated at 37 °C for 15 minutes. The suspension was transferred to a Lonza Nucleocuvette™ and electroporated with the CA-137 program code. Cells were suspended in growth media containing 10 pM ROCK inhibitor and transferred to a 24-well plate. The medium was exchanged the following day. Single cell populations were obtaining using limited dilution and genetic knockouts were confirmed by Synthego ICE CRISPR analysis and western blot.

[00236] Generation and Purification ofGPG, GPE, and GPS Phosphatidylglycerol (16:0), lysophosphatidylethanolamine (18:1), and lysophosphatidylserine (18:1) (Avanti) were saponified to produce GPG, GPE, and GPS as follows: To a 3 mL 2:1 chloroform:methanol solution in a 20 mL scintillation vial containing a magnetic stirring bar, 25 mg of lipid was added and dissolved by vortexing. 1 mL 2 M NaOH was added to the mixture, stirred at room temperature for 2 hours, and quenched with 1 mL 2 M HC1. 2 mL water was used to extract GPDs twice into separate tubes. Solutions were combined and washed with 2 mL chloroform. The GPD-containing aqueous phase was lyophilized and dissolved in methanol to precipitate inorganic salts. Pure GPDs were separated from precipitants by paper filtration.

[00237] Immunoblotting Lysates were ran on an 8-16% SDS-PAGE (Thermo Fisher Scientific) at 120 V. Proteins were transferred onto nitrocellulose membranes for 1 hour 30 minutes at 100 V. Membranes were blocked with Intercept (TBS) Blocking Buffer (LICOR) for 1 hour and incubated with primary antibodies in TBS Blocking buffer overnight at 4°C. Membranes were then washed with TBST (Tris-buffered saline with Tween 20) 3 times for 5 minutes and then incubated with secondary antibodies diluted 1:15,000 in TBS Blocking Buffer for 1 hour at room temperature. Membranes were then washed 3 times with TBST and visualized using fluorescence imaging.

[00238] BMPS Protein Expression and Purification Human CLN5 was cloned into a pRK5 plasmid with a C-terminal 6xHis tag preceded by a short linker sequence (GGGGS (SEQ ID NO: 11)) respectively. Expi293F cells grown in a 1/3 Expi293 media and 2/3 Freestyle293 media combination were transfected with plasmids at a density of 3E6 cells/mL and cultured in a shaking incubator at 37°C and 8% CO2. Transfection was performed using the FectoPRO transfection reagent (Polyplus) at ratios of 1.3uL FectoPRO and 0.5ug plasmid DNA per mL of cells. Immediately after transfection, cells were boosted with glucose and valproic acid to final concentrations of 0.4% glucose (w/v) and 0.05% valproic acid (w/v). One day post-transfection, cells were again boosted with an equivalent amount of glucose and valproic acid. Cells were harvested three days post-transfection.

[00239] The media was collected, diluted 1:1 in 1XPBS (Phosphate buffered saline), and incubated with HisPur Ni-NTA resin (Thermo Fisher) for 16 hours at 4°C. The resin was then washed with four iterative washes (wash 1 composition: 50 mM HEPES pH 7.25, 500 mM NaCl, 0.1 mM EDTA, 5 mM beta-mercaptoethanol, 5% glycerol [v/v], 20 mM imidazole pH 7.25, 1 mM DTT, 1 mM PMSF, cOmplete EDTA-free protease inhibitor cocktail (Roche), and 1% Triton X-100 [v/v]; wash 2 composition: same as wash 1 composition without Triton X-100 and protease inhibitor and with 10 mM imidazole pH 7.25; wash 3 composition: same as wash 2 without imidazole and with 250 mM NaCl; wash 4 composition: same as wash 3 with 125 mM NaCl). The resin was packed onto a column and protein was eluted in a buffer containing 300 mM Imidazole pH 7.25, 50 mM HEPES pH 7.25, 125 mM NaCl, 5 mM BME, 5% glycerol [v/v], 1 mM DTT, 1 mM PMSF, 0.1 mM EDTA, and protease inhibitors. Eluted protein was concentrated using an Amicon 10-kDa MWCO concentrator. Purified protein fractions were collected after size-exclusion chromatography (SEC) over a Superdex 200 10/300 column in a buffer of 50 mM Tris pH 6.8 and 150 mM NaCl. The fractions were pooled, concentrated to a final BMPS concentrations of 1 mg/mL using an Amicon 10-kDa MWCO concentrator, and flash-frozen in liquid nitrogen.

[00240] Microscale Thermophoresis Purified human BMPS -6xHis WT, K319E, & HK318/9EE were labeled with the RED-NHS Protein Label Kit (Nanotemper Technologies) as directed by the manufacturer’s protocol, and experiments were performed on a Monolith NT.l 15 instrument (Nanotemper Technologies). Labeled BMPS (100 nM) was incubated with 2:1 serial dilutions of substrate in 50 mM Sodium Acetate-Acetic Acid pH 5.5 or 50 mM HEPES pH 7.5, 150 mM NaCl, and 5 pM BSA for 30 minutes at room temperature. Binding experiments were performed at 20%, 40%, or 60% MST power for 30 s with 5 s of cooling using premium capillaries (Nanotemper Technologies). Dissociation constant K& was obtained by plotting the fraction bound against logarithmic substrate concentrations. In cases where no binding was detected, Fnorm was plotted against logarithmic substrate concentrations instead.

[00241] Thermal Stability Assays Experiments were performed on a Tycho NT.6 instrument (Nanotemper Technologies). Purified human BMPS WT and mutants were diluted to 5 pM in 50 mM Sodium Acetate- Acetic Acid pH 5.5 and 150 mM NaCl and loaded into Tycho NT.6 capillaries. Melting temperature T_m was obtained from plotting the absorbance ratio of solvent exposed tryptophan to buried tryptophan (350 nm/330 nm) over a temperature gradient and determining the inflection temperature from the first derivative. Melting temperatures were obtained as indicated above.

[00242] Circular Dichroism Spectroscopy All circular dichroism (CD) spectra were obtained on a J-815 spectrometer (Jasco). Purified human BMPS WT and mutants were diluted to 6 pM (0.25 mg/mL) in 50 mM Tris pH 6.8 and 150 mM NaCl in a 1.0 mm quartz cuvette at room temperature. Three accumulations were obtained for the far UV spectra (200 nm-260 nm) and averaged with 50 nm/min scanning speed, 1 .00 nm band width, 0.1 nm step size, and 1 sec integration time.

[00243] Isolation of lysosomes Lysosomes were isolated and metabolically profiled as described in Abu-Remaileh et al. 2017 (Science. 358, 807-813, incorporated herein by reference in its entirety). Briefly, cells were infected with TMEM192-3xHA lentivirus for lysosome immunoprecipitations and seeded on 15 cm plates at confluency. Cells were washed with KPBS (136 mM KCL, 10 mM KH2PO4, pH 7.25 in Optima LC-MS water), lifted, and dounce homogenized to release subcellular compartments. HA-tagged lysosomes were bound by magnetic anti-HA beads for five minutes, washed three times with KPBS, and lysed in a 2:1 chloroforrmmethanol solution (v/v) with a 750 ng/mL SPLASH LIPIDOMIX internal standard mix (Avanti).

[00244] Lipid and Metabolite Extraction Lipids were extracted for 1 hour, and further extracted after the addition of a 0.9% saline solution for 10 minutes. The chloroform phase was removed, concentrated in vacuo, reconstituted in a 13:6:1 acetonitrile:isopropanol:water (v/v/v) solution, and stored at -80C until analysis by LC-MS/MS. Metabolites were harvested in 80% methanol with isotopically labeled amino acids and stored at -80C until analysis by LC-MS/MS. [00245] Data preparation and statistics All quantitative graphs and one site specific binding curves were generated in GraphPad Prism 9. Two-tailed independent t-tests and ordinary one- ANOVA for statistical comparisons were calculated in Prism. Fold changes for normalized lipid intensities were calculated by baseline correction in GraphPad Prism 9. All measurements represent samples generated by biological replicates.

[00246] Schrodinger Docking A published crystal structure of BMPS (PDB 6R99, Chain A) was loaded into Maestro software (Schrodinger Release 2020-2). Protein was prepared for docking using the protein preparation wizard to add hydrogens, create disulfide bonds, assign bond orders, delete waters beyond 5 A of het groups, fill in missing sides chains using Prime, and cap termini. Ionization and tautomeric states were generated at pH 5 +/- 0.5 to reflect lysosomal pH. Furthermore, Hydrogen-bonding assignment was optimized at pH 5 and set to sample water orientations. Waters with fewer than three hydrogen bonds to non-waters were removed. Restrained minimization was performed using the OPLS3e force field. Ramachandran plots were used to inspect steric clashes. Receptor grids were generated without a ligand and enclosed the entire protein with appropriate x, y, & z coordinates. 3D 18:1 LPG libraries were generated using the LigPrep wizard. Specifically, a 2D 18: 1 LPG structure was drawn in ChcmDraw, and LigPrep was used to generate possible states at target pH 5 +/- 0.5 via Epik, tautomers, and stereoisomers in an OPLS3e force field. For docking the LPG library into BMPS, the extra precision (XP) docking mode of Glide was used under the following parameters: flexible ligand sampling, add Epik state penalties to docking score, keep 100% of scoring compounds, default van der Waals Radii scaling, and perform post-docking minimization. [00247] Liposome Preparation For liposomes without lysophospholipid, lipids were dried under vacuum and resuspended in 3 mL diisopropyl ether and 2 mL water in a 5 mL eppendorf tube. The organic, aqueous solution was subsequently sonicated in a water bath for 10 minutes to form small unilamellar vesicles and dried under vacuum. Dried liposomes were resuspended in water, and their concentration was determined by Stewart assay. For liposomes with lysophospholipid, lipids were dried under vacuum and resuspended in a volume of water corresponding to the desired assay stock concentration. The aqueous suspension was subsequently sonicated in a water bath for 10 minutes to disperse lipid aggregates and used directly in assays.

[00248] BMP synthesis assay 100 nM BMPS-6xHis and mutants were incubated with indicated lysophospholipid substrate concentrations in 50 mM Sodium Acetate:Acetic Acid pH 5.0 and 150 mM NaCl for 1 minute at 37 °C and heat-inactivated at 95 °C for 3 minutes. Care was taken to exclude lipid-binding carrier proteins and nonionic detergents. All lysophospholipid substrates were added below their critical micellar concentrations. To monitor glycerophosphodiester release, reactions were directly transferred to plastic autosampler vials. To monitor bis(monoacylglycero)phosphate release, reactions were diluted with an equal volume of acetonitrile and transferred to glass autosampler vials.

[00249] BMPS pH Optimum Buffers were used at a 50 mM final concentrated and prepared using Sodium Acetate: Acetic Acid, HEPES, and Boric acid.

[00250] Enzyme Replacement To validate the lysosomal localization of recombinant enzymes, BMPS-6xHis and mutants were labeled with NHS-Alexa488 dye (Thermo Fisher Scientific) according to the manufacturer’s protocol. Afterwards, 100,000 cells were seeded onto a four- chamber 35 mm dish in complete media. After 24 hours, cells were treated with 50 nM fluorescently labeled protein in complete media for 24 hours. Cells were washed once with PBS and incubated in complete media without phenol red containing 37.5 nM LysoTracker Red DND-99 and 10 ug/mL Hoechst 33342. Cells were imaged on a ZEISS LSM 980 confocal microscope. Images were analyzed using ImagcJ. For enzyme replacement experiments, 600,000 cells were seeded on a 6- well plate. After 24 hours, cells were treated with 50 nM fluorescently labeled protein in complete media. After 72 hours, lipids were extracted and analyzed by lipidomics.

[00251] Deuterated Lipid Tracing A 10X solution of deuterated lipid tracers d5-PG (16:0/18:1) and d9-PC (18:0/18:0) at 10 M in ethanol and of fatty acid free BSA at 10 pM in PBS were prepared in DMEM (ThermoFisher) and diluted to a IX solution in DMEM. Cells starved in serum-free DMEM for two hours were fed the IX BSA-conjugated lipid tracer solution at different time points, and metabolites and lipids were harvested via two-phase extraction in 80% methanol and/or 2:1 chloroforrmmethanol (v/v).

[00252] Untargeted lipidomics workflow Profiling of nonpolar lipids was performed on an ID- X Tribrid mass spectrometer (Thermo Fisher Scientific) with a heated electrospray ionization (HESI) probe. An Ascentis Express C18 150 x 2.1 mm column (Millipore Sigma 53825-U) coupled with a 5 x 2.1 mm guard (Sigma-Aldrich 53500-U) was used to carry out C18-based lipid separation prior to mass spectrometry. Mobile phases: A, 10 mM ammonium formate and 0.1% formic acid dissolved in 60% and 40% LC/MS grade water and acetonitrile, respectively; B, 10 mM ammonium formate and 0.1% formic acid dissolved in 90% and 10% LC/MS grade 2- propanol and acetonitrile, respectively. Chromatographic gradient: isocratic elution at 32% B from 0—1.5 minutes; linear increase from 32-45% B from 1.5-4 minutes; linear increase from 45- 52% B from 4-5 minutes; linear increase from 52-58% B from 5-8 minutes; linear increase from 58-66% B from 8-11 minutes; linear increase from 66-70% B from 11-14 minutes; linear increase from 70-75% B from 14-18 minutes; linear increase from 75-97% B from 18-21 minutes; hold at 97% B from 21-35 minutes; linear decrease from 97-32% B from 35-35.1 minutes; hold at 32% B from 35.1-40 minutes. Flow rate, 0.26 ml/minutes. Injection volume, 2-4 pL. Column temperature, 55°C. Mass spectrometer parameters: ion transfer tube temperature, 300 °C; vaporizer temperature, 375 °C; Orbitrap resolution MSI, 120,000, MS2, 30,000; RF lens, 40%; maximum injection time MSI, 50 ms, MS2, 54 ms; AGC target MSI, 4x105, MS2, 5x104; positive ion voltage, 3250 V; negative ion voltage, 3000 V; Aux gas, 10 units; sheath gas, 40 units; sweep gas, 1 unit. HCD fragmentation, stepped 15%, 25%, 35%; data-dependent tandem mass spectrometry (ddMS2) cycle time, 1.5 s; isolation window, 1 m/z; microscans, 1 unit; intensity threshold, 1.0e4; dynamic exclusion time, 2.5 s; isotope exclusion, enable. Full scan mode with ddMS2 at m/z 250-1500 was performed. EASYICTM was used for internal calibration. LipidSearch and Compound Discoverer (Thermo Fisher Scientific) were used for unbiased differential analysis. Lipid annotation was acquired from LipidSearch with the precursor tolerance at 5 ppm and product tolerance at 8 ppm. The mass list was then exported and used in Compound Discoverer for improved alignment and quantitation. Mass tolerance, 10 ppm; minimum and maximum precursor mass, 0-5,000 Da; retention time limit, 0.1-30 min; Peak filter signal to noise ratio, 1.5; retention time alignment maximum shift, 1 min; minimum peak intensity, 10,000; compound detection signal to noise ratio, 3. Isotope and adduct settings were kept at default values. Gap filling and background filtering were performed by default settings. The MassList Search was customized with 5 ppm mass tolerance and 1 minute retention time tolerance. Area normalization was performed by constant median after blank exclusion. [00253] Targeted Lipid and Metabolite Quantitation Lipids were separated on an Ascentis C18 column (5 Micron, 5 mum particle size, L x I.D.5cm x 4,6mm) (Sigma-Aldrich) with an Ascentis Express guard holder and connected to a 1290 LC system. A 6470A triple quadrupole (QQQ) mass analyzer equipped with an LC-ESI probe was coupled to the LC system. An external mass calibration was performed using the standard calibration mixture every 7 days, and injection volumes of 4 pL were used for each sample with fast polarity switching. Mobile phase A was composed of 10 mM ammonium formate and 0.1% formic acid in LC/MS grade 60:40 water: acetonitrile with 10 mM ammonium formate. Mobile phase B was composed of 10 mM ammonium formate and 0.1% formic acid in 90:10 isopropanokacctonitrilc. The chromatographic gradient was the following: isocratic elution from 0-1 minutes with 32% B, linear increase from 32-66% B from 1-6 minutes; linear increase from 66-75% B from 6-10 minutes; linear increase from 75-97% B from 10-14 minutes; fast linear decrease from 97-32% B from 14-14.5 minutes; 32% B hold from 14.5-18.5 minutes. The flow rate was set to 0.260 mL/min, and the column compressor and autosampler were held at 55 °C and 4 °C, respectively. [00254] Metabolites were separated on a Sequant HILIC column (5 micron particle size, I.D. x L = 2.1 x 150 mm) (Millipore Sigma) with a Sequant column guard and connected to a 1290 LC system. Injection volumes of 2.5 uL were used for each sample with fast polarity switching. Mobile phase A was composed of 20 mM ammonium carbonate and 0.1% ammonium hydroxide dissolved in 100% LC/MS grade water. Mobile phase B was composed of 100% LC/MS grade acetonitrile. The chromatographic gradient was the following: linear decrease from 80-20% B from 0-7 minutes; fast linear increase from 20-80% from 7-7.5 minutes; 80% hold from 7.5-10 minutes. The flow rate was set to 0.150 mL/min, and the column compressor and autosampler were held at 55 °C and 4 °C, respectively.

[00255] The mass spectrometer parameters were as follows: the spray voltage was set to 3.5 kV in positive mode and 2.5 kV in negative mode, and the gas temperature and the sheath gas flow were held at 250 °C and 300 °C, respectively. Both gas flow and sheath gas flow were 12 L/min while the nebulizer was maintained at 25 psi. The mass spectrometer was operated in Multiple Reaction Method (MRM) for targeted analysis of species of interest.

[00256] Standard compounds including d5-PG (16:0/18:1), d9-PC (18:0/18:0), LPG (18:1), LPC (18:1) and PC (16:0/18:1) (POPC) were purchased from Avanti Polar Lipids; GPC from Sigma; GPI from Echelon Biosciences; GPG, GPE, and GPS chemically synthesized in house. These standards were optimized using a MassHunter Optimizer MRM. MassHunter Optimizer MRM is an automated method development software used to generate and optimize MRM transitions accumulating at most 4 products with different abundances from singly ionized species. The two most abundant transitions from either the negative or positive mode were selected to detect each species.

[00257] The precursor-product ion pairs (m/z) used for MRM of the compounds were the following:

[00258] High-throughput annotation and relative quantification of lipids were performed using a qualitative analysis software of MassHunter acquisition data and QQQ quantitative analysis (Quant-My-Way) software. Individual lipid species shown in the figures were validated using the Qualitative software by manually checking the peak alignment and matching the retention times and MS/MS spectra to the characteristic fragmentation compared to the standard compounds. Analyzing two transitions for the same compound and looking for similar relative response was an added validation criterion to ensure the correct species were identified and quantified. The MRM method and retention time were used to quantify all lipid species using the quantification software, and the raw peak areas of all species were exported to Microsoft Excel for further analysis. Raw abundances were normalized to cell number using abundance of endogenous control lipids in the same sample.

[00259] Filipin Staining Cells (10,000) were seeded onto 8-well tissue culture slides coated with fibronectin and treated with recombinant BMPS (50 nM) for 48 hours. Cells were rinsed with PBS three times, fixed with paraformaldehyde, and stained with 0.05 mg/mL Filipin III (Cayman) in 1% BSA in PBS for two hours at room temperature. Cells were rinsed with PBS three times, mounted, and imaged on a Zeiss LSM 980 confocal microscope. Images were analyzed using ImagcJ.

[00260] BMP Synthesis in Lysosomal Protein Extract Cells were seeded onto 15 cm plates at confluency. Lysosomes were immunoprecipitated using the above described methodology and hypotonically lysed in water for 30 minutes to release soluble lysosome proteins. Lysosomal protein extract was incubated with 10 pM d5-PG (16:0/18:1) tracer and 1 pM recombinant BMPS in 50 mM Sodium Acetate: Acetic Acid pH 5.0 and 150 mM NaCl for 2 hours at 37 °C, and the reaction was quenched with a 2:1 chloroform: methanol (v/v) solution.

[00261] Multiple Sequence Alignment Primary sequences for human (077503), mouse (Q3UMW8), bovine (Q1ZYR0), sheep (A2TJ54), and dog (Q5JZQ9) BMPS were obtained from UniProt, and sequence alignment was performed with CLUSTAL Omega.

Example 1 CLN5 loss reduces BMP

[00262] Untargeted lipidomics was used to analyze the lipidome of CLN5 knockout HEK293T lysosomes (FIG. 1A). With MSI and tandem mass spectrometry (MS/MS) fragmentation data alone, a massive accumulation of lysophosphatidylglycerol (LPG), a detergent-like lysophospholipid, was found (FIG. 1A). The acyl composition of LPG did not affect the accumulation phenotype, suggesting the entire LPG class is uniformly aberrant (FIG.

1 A). Intriguingly, a marked depletion of a glycerophospholipid species consistent with BMP was also observed (FIG. 1A). This analysis revealed that BMP species are uniformly depleted in CLN5 knockout HEK293Ts (FIG. IB). Importantly, the levels of phosphatidylcholine (PC) were unchanged, and thus were used for normalization (FIG. 5B). Notably, ceramides were slightly deficient (FIG. 1A). Given the alterations in LPG and BMP, their levels in CLN5 knockout HEK293Ts were quantified at the whole-cell and lysosomal level using optimized multiple reaction monitoring (MRM) transitions for each class. This targeted analysis confirmed LPG accumulation and BMP deficiency (FIGS. IB and 1C). Consistent with BMP’s localization to LE/LY, whole cells were comparably deficient in BMP and exhibited similar LPG storage (FIGS. IB and 1C).

[00263] To test whether CLN5 loss of function affects BMP homeostasis in physiologically relevant systems, CLN5-deficient human induced pluripotent stem cells (iPSCs) were generated and differentiated into neurons (iNeurons) (FIG. 5D). Consistent with results in CLN5 knockout HEK293Ts, targeted quantitation of BMP and LPG in CLN -deficient iPSCs and iNeurons revealed substantial storage of LPG and depletion of BMP species at the whole-cell level (FIG. ID). Thus, CLN5 loss of function depletes BMP levels.

Example 2 CLN5 as BMP Synthase (BMPS)

[00264] Recombinant, His-tagged CLN5 protein was expressed and purified to homogeneity as a multimeric protein (FIGS. 6A-6B, and 6F-6G ), which possessed weak thioesterase activity (FIG. 6H). Taking care to be below the critical micellar concentration of LPG (18:1), recombinant CLN5 protein was incubated with LPG (18:1) at acidic pH and the reaction was allowed to proceed for fifteen minutes. Two-phase extraction and MS/MS quantitation of LPG (18:1), BMP (18:1/18:1), and its predicted byproduct glycerophosphoglycerol (GPG) revealed the presence of BMP (18:1/18:1) and GPG only in conditions containing CLN5 protein and LPG (FIGS. 2A- 2C). Importantly, the glycerophospholipid was validated as BMP (18:1/18:1), as a commercially available standard had an identical retention time and MS/MS spectrum (FIG. 2D). Therefore, herein CLN5 protein, or the protein encoded by CLN5, is also referred to as BMP synthase (BMPS). A kinetic enzyme assay of BMPS activity against LPG monitoring was performed for BMP and GPG release and moderate enzyme activity (k_Cat/K_m = ~10⁴ M'¹ s'¹) was observed for both products (FIGS. 2E and FIG. 7A). As a result, the kinetic quantitation of GPG product was used for all further BMPS assays. Notably, removal of the polyhistidine tag did not alter BMPS activity (FIG. 7D). Consistent with its LE/LY localization, BMPS synthesized BMP at a slightly acidic optimum of 6.5 with a catalytic efficiency of ~10⁵ M^{- I}s^_| although appreciable activity is still retained at more acidic pH (FIG. 7B and 7E).

[00265] To rule out exclusive activity against LPG (18:1), BMPS activity was tested against various LPG species. BMPS displayed activity against all tested LPGs (LPG 14:0, LPG 16:0, LPG 18:0, LPG 18:1), with a higher preference for longer chain lengths (FIG. 7C).

[00266] BMPS N143S is a patient missense mutation known to retain protein folding and lysosome trafficking. Consistent with this literature, no alterations in recombinant BMPS N143S secondary structure, conformation, and thermal stability were observed relative to the wild type (WT) (FIG. 61, 11C, and 12C). Still, BMPS N143S possessed considerably weakened enzyme activity (FIG. 20A), suggesting that defective BMP synthesis may drive CLN5 Batten disease. [00267] To establish a system to evaluate the LE/LY role of BMPS activity in modulating BMP and LPG within cells, given that several studies support a role of BMPS in cxtralysosomal secretory compartments like the endoplasmic reticulum (ER) and Golgi, conditioned media was supplemented with fluorescently labeled BMPS for endocytic delivery to CLN5 knockout cells and its localization to LE/LYs was confirmed by fluorescence microscopy (FIGS. 8A-8D and 21 A-21F). With this important control, it was tested whether recombinant BMPS replacement in CLN5 knockout cells can normalize their aberrant lipid profile (FIG. 2F). Recombinant BMPS protein replacement in CLN5 knockout cells for only 48 hours rescued LPG accumulation and significantly restored BMP levels (FIGS. 2G, 9A and 9B). Supplementation of CLN5 -deficient cells with recombinant BMPS N143S protein failed to restore LPG and BMP homeostasis (FIGS. 20B-20C and 21A-21F). These data demonstrate BMPS localization to the ER or Golgi is not required for BMPS activity in cells and the CLN5 gene product is the lysosomal BMP synthase. [00268] As it relates to NCL, disruption of BMPS activity and thus BMP synthesis is a common phenotype across genotypes that define this lysosomal storage disease. The levels of BMP in NCL patients have also been shown to be disrupted in multiple genotypes including as a result of mutations to CLN2 and CLN3 (Hobert et al. Biochem. Biophys. Res. Commun. 358:111- 6 (2007);Tessa et al. Human M t. Brief 238 (2000)). Given genetic mutations within the NCL family relate to BMP phenotypes, it is expected that NCL patients across all genotypes would benefit from a therapeutic that could correct BMP levels and normalize lysosomal function, and this could include a BMPS therapeutic.

[00269] Therapeutic benefit from BMPS dosing could then extend to other lysosomal storage diseases (LSDs) as well given the established requirement of BMP for breakdown of lipids that accumulate and become toxic in a wide range of LSDs, such as Gaucher’s disease (caused by inability to breakdown glucosylceramide), Tay-Sach’s disease (caused by inability to breakdown GM2 ganglioside), metachromatic leukodystrophy (caused by inability to breakdown sulfated glycosphingolipids), Fabry (caused by inability to breakdown globotriaosylceramide), GM1 (caused by inability to breakdown GM1 ganglioside), Krabbe (caused by inability to breakdown galactosylceramide), Niemann Pick Type A/B (caused by inability to degrade sphingomyelin), and Sandhoff (caused by inability to breakdown GM2 ganglioside) (Rouser et al. Lipids 3:287-90 (1968); Miekle et al. Biochem. J. 411(1):71-8 (2008); Abe et al. J Lipid Res. 50:2027-35 (2009); Wilkening et al. J Biol Chem. 273:30271-78 (1998).; Linke et al. Biol Chem. 382:283-90 (2001); Werth et al. J Biol Chem. 276:12685-90 (2001)). Consistently, Bmps KO and Grn (Clnl 1 ) KO cells have reduced capacity to breakdown glucosylccramidc, and BMPS dosing rescues this phenotype. Taken together, these data strongly support the potential for a BMPS therapeutic to translate into a disease-modifying therapy for a spectrum of lysosomal storage diseases .

Example 3

BMP laden vesicles enhance BMP synthesis

[00270] The activation of lipid enzymatic activity through the electrostatic attraction of cationic lysosomal enzymes onto negatively charged ILVs is a central dogma in lysosomal lipid catabolism. BMP imbues ILVs with a net negative charge. BMPS could only interact with liposomes containing both phosphatidylcholine and BMP and required an acidic pH for docking (FIGS. 2H and 10A).

[00271] Polyhistidine tags have been shown to mediate binding to BMP liposomes. To rule out a polyhistidine tag-mediated interaction between BMPS and BMP liposomes, the BMPS motif responsible for docking was investigated. Whether the BMPS cationic amphipathic helix (CAH) mediates docking onto ILVs through its well-conserved cationic residues was investigated (FIGS. 10B and 10C). Consistent with the hypothesis, mutation of those residues to negatively charged glutamates did not alter the properties or stability of the protein (FIGS 6C, 6D, 11A, and 12A), but weakened vesicular BMP binding (FIG. 10D).

[00272] To test for the stimulation of BMPS activity by BMP liposomes, LPG (18:1) was integrated into liposomes at a constant lysophospholipid to liposome equimolar ratio. This is important to maintain stimulation of activity and avoid surface dilution (FIG. 13A). Expectedly, BMP liposomes biophysically enhanced BMP synthesis consistent with a previous study (FIG. 21) and a surface dilution effect was observed with higher liposome to substrate ratios consistent with lipid enzyme theory (FIG. 13B). Consistent with studies on the ability of other anionic phospholipids to stimulate lysosomal lipid enzymes, increased BMPS activity was observed on membranes containing PG, phosphatidylinositol, and phosphatidylserine, although the relatively high abundance of BMP in ILVs suggested BMP acts as the main anionic coactivator in lysosomes (FIG. 13C).

[00273] Amiodarone, a cationic amphiphilic drugs (CAD) shown to cause drug-induced phospholipidosis, slightly inhibited BMPS activity towards non-liposomal LPG and dramatically inhibi ted BMPS activity towards liposomal LPG likely through neutralization of the negative charge on the surface of BMP liposomes (FIGS. 2J and 2K).

Example 4 A base-exchange reaction mediates BMP synthesis

[00274] If a base-exchange reaction between two LPG molecules mediates BMP synthesis it would result in an initial release of glycerolphosphoglycerol during LPG deacylation without any energy input. Consistently, the water-soluble GPG byproduct was detected along with BMP, supporting this mechanism (FIG. 2C). In principle, the base-exchange reaction could utilize any lysophospholipid as an acyl donor, so it was asked whether other lysophospholipids, namely lysophosphatidylcholine (LPC), lysophosphatidylserine (LPS), lysophosphatidylethanolamine (LPE), and lysophosphatidylinositol (LPI), are additional BMPS substrates. No appreciable release of glycerophosphocholine (GPC), glycerophosphoserine (GPS), glycerophosphoethanolamine (GPE), or glycerophosphoinositol (GPI) was detected suggesting these LPGs are not substrates for BMPS (FIG. 14A). Consistent with these findings, LPC, LPE, and LPS do not accumulate in BMPS-deficient lysosomes, and LPI only modestly increases FIGS. 14B-E). Thus, LPG represents the major lysophospholipid acyl donor for base-exchange. [00275] To identify the BMPS active site, in silico docking experiments identified a plausible active site that positioned the LPG carbonyl carbon near cysteine 231, histidine 117, & glutamate 134 catalytic triad (FIG. 3A). Because the predicted thioester enzyme-acyl intermediate represents a stronger electrophile than its ester counterpart for base-exchange with LPG, whether a cysteine-to-serine BMPS substitution attenuated enzyme activity was investigated. C231S BMPS mutation did not alter the properties or stability of the protein (FIGS 6E, 1 IB, and 12B). C231S BMPS retained less than 5% of the wildtype BMPS activity (FIGS. 3B and 14F), and its replacement in CLN5 knockout cells for 48 hours partially rescued LPG accumulation and BMP deficiency relative to that of wildtype BMPS (FIGS. 3C, 3D, 14G and 14H). These data support an energy-independent, active site thiol mediated base-exchange mechanism for BMP synthesis (FIG. 3E).

Example 5 Late endosomes/lysosomes require BMPS to synthesize BMP

[00276] Given the localization of BMPS to lysosomes and lysosomal deficiency of BMP, an established method was used to conjugate lipid tracers to bovine serum albumin (BSA) for efficient endocytic internalization and delivery (FIG. 4A). CLN5 knockout cells were fed a BSA- conjugatcd, dcutcrium-labclcd (d5-)PG (16:0/18:1) tracer for various time points and conversion of PG to LPG and BMP was assessed in LE/LYs by monitoring for the appearance of deuterium- labeled metabolites (FIG. 4B). CLN5 knockout cells converted d5-PG (16:0/18:1) to d5-LPG (18:1) with comparable kinetics to wildtype (WT), but exhibited a complete block in d5-LPG (18:1) conversion to d5-BMP (18:1/18:1) (FIG. 4C and 15A). CLN5 knockout cells accumulated more d5-PG (16:0/18:1) over time compared to WT, indicating either reduced turnover or faster endocytic uptake (FIG. 15B). These data demonstrated that LE/LYs of CLN5 knockout cells are unable to transform LPG into BMP consistent with the molecular function of BMPS to mediate transacylation of LPG to form BMP. Although these data support LE/LYs as the site of BMP synthesis, to evaluate LE/LY-mediated BMP synthesis in vitro, BMPS-deficient LE/LYs of were immunoprecipitated and assessed for their ability to convert deuterated PG to BMP (FIG. 4D). Indeed, BMPS deficient lysosomal lysates were unable to convert d5-PG(16:0/18:l) to d5-BMP (18:1/18:1) (FIG. 4E). Complementation of BMPS deficient lysosomal lysate with recombinant CLN5 protein restored synthesis of d5-BMP (18:1/18: 1) and d5-BMP (16:0/18:1) (FIG. 4E).

[00277] CLN5 knockout cells fed a BSA-conjugated d9-PC (18:0/18:0) tracer for various timepoints displayed reduced kinetic conversion of d9-PC (18:0/18:0) to d9-LPC (18:0), suggesting weakened phospholipase activity (FIGS. 4F and 4G). Of importance, endocytic uptake of d9-PC (18:0/18:0) was similar between wildtype and CLN5 knockout cells (FIG. 15C). [00278] CLN5 -deficient HEK293Ts exhibited slower conversion of d5-GlclCer (dl 8: 1/18:0) to d5-Cer (dl8: 1/18:0) without reduced endocytic uptake, indicating attenuated GCase (Glucocerebrosidase) activity (FIGS. 41, 4J, and 15D). CLN5 knockout HEK293T cells, iPSCs, and iNeurons did not exhibit an increase in the levels of hexosylceramides (FIG. 22A) although CLN5 knockout HEK293T cells and iPSCs were deficient in ceramides, which were largely unaffected in iNeurons (FIG. 22B). Still, CLN5 knockout iNeurons accumulated GM3 gangliosides (FIG. 4K), another lipid whose degradation is dependent on BMP. Notably, such gangliosidosis is known to drive neurodegeneration.

[00279] Furthermore, filipin staining of CLN5 knockout cells revealed excess cholesterol storage (FIG. 4H). Importantly, treatment of CLN5 knockout cells with recombinant CLN5 protein rescued secondary cholesterol storage material consistent with the role of BMP in maintaining cholesterol homeostasis (FIG. 4H). Example 6 Characterization of glycerophosphodiester (GPD) inhibition of BMPS

[00280] A thermal shift assay (TSA) was performed with recombinant BMPS (rBMPS) and GPDs. Incubation of rBMPS with GPG and GPS resulted in increased thermal stability while glycerol control had only a marginal effect (FIG. 17A). To confirm direct binding, the strength of the rBMPS:GPD complex was determined by MST and it was found that rBMPS binds GPS, GPE, GPI, and GPC with micromolar affinity (FIG. 17B). rBMPS was unable to degrade GPE (FIG. 17C).

[00281] GPG was incubated with rBMPS and BMP synthesis activity was assessed. GPG potently inhibits rBMPS (FIG. 18A). Incubation of rBMPS with other glycerophosphodiesters (GPE, GPS, GPC, and GPI) likewise inhibited BMP production (FIG. 18B).

[00282] The Michaelis-Menten inhibition curve suggested that GPDs inhibit BMPS at an allosteric site (FIG. 18A). In silico modeling identified a plausible basic pocket on BMPS for GPD binding and mutations with this pocket, namely K296A, K73A, & N143S, each attenuated binding, with K73A and K296A showing the strongest attenuation of binding (FIGS. 19A and 19B). BMPS K296A retained some BMP synthesis activity (FIG. 19C). Additionally, incubation of BMPS K296A with GPG no longer resulted in inhibition (FIG. 19D). These data demonstrate GPDs bind to BMPS with a micromolar Ka, inhibit BMP synthesis activity, likely binding at an allosteric site comprising K296, K73, & N143.

Example 7

Exogenous BMPS addition rescues Niemann-Pick Type C disease phenotype in vitro [00283] A frozen vial of fibroblasts derived from a healthy patient (Coriell # GM05659) or NPC1 patient (Coriell # GM 18453) were purchased from Coriell. Frozen vials were thawed quickly in a 37 °C water bath and added to 10 mL of complete DMEM medium supplemented with 10% FBS, 100 I.U./mL penicillin, and 100 pg/mL streptomycin. Fibroblasts were routinely cultured in complete DMEM and split at a 1 to 2 ratio after reaching 80-90% confluence to maintain the cell in culture. Fibroblasts (50k) from either healthy or NPC1 patient lines were seeded on an 8-Chamber cell culture slide (Celltreat) containing 500 pL complete DMEM, subsequently treated with vehicle (1XPBS) or 150 nM of BMPS, and left to incubate overnight at 37 °C. Afterwards, the medium was gently aspirated and cells were washed once with 800 pL 1XPBS, followed by immediate 15 min fixation by the addition of 200 pL of a 4% PFA solution in 1XPBS . The cells were then washed once with 800 pL of 1XPBS, stained with 200 pL of 1 mg/ml Filipin (Cayman), scaled with glass coverslip, scaled with mounting solution (Thermo Scientific), and imaged with Leica LSM98O confocal microscope. One-way ANOVA was performed by Prism.

[00284] As shown in FIG. 23, treatment with BMPS reduce filipin intensity per cell.

[00285] Filipin is a well precedented tool for staining and monitoring cholesterol distribution and accumulation in vitro and in cellulo. The reduction of filipin intensity per cell after BMPS treatment in NPC patient-derived cells signifies a reduction in cholesterol content within the cells, correcting the significant cholesterol accumulation phenotype that is the hallmark and clinically defining phenotype of Niemann-Pick Type C disease (NPC). In particular, NPC disease is driven by cholesterol accumulation in lysosomes, and treatments that lower cholesterol, such as cyclodextrins, are known to be therapeutically efficacious. Therefore, the reduction of cholesterol storage in NPC patient cells via dosing of recombinant BMPS demonstrates the potential for a BMPS-targeting therapeutic to translate into a therapy for NPC and other lysosomal storage diseases.

[00286] The therapeutic benefit of the BMPS-mediated reduction of cholesterol in lysosomes extends to other diseases with aberrant cholesterol storage. For example, in Alzheimer’s disease (AD), mutations to apolipoprotein E (APOE), the strongest risk genetic factor for AD, impair myelination in oligodendrocytes due to cholesterol dyshomeostasis (Blanchard et al. Nature 611:769-79 (2022)). Such cholesterol dyshomeostasis in models of AD can be rescued with cyclodextrins in highly comparable fashion to the correction of the cholesterol phenotypes describe in NPC above (Blanchard et al. Nature 611:769-79 (2022); Miranda et al. Transl. Psychiat. 12:129 (2022); Sienski et al. Sci. Transl. Med. 13(583):eaaz4564 (2021); Cruchaga et al. Res. Sq. (2023) doi:10.21203/rs.3.rs-2814616/vl). Additionally, mutations to the BMPS gene (CLN5) can cause a rare inherited form of Alzheimer’s disease (Qureshi et al. Mol. Cell. Biol. 38(20):e00011-18 (2018)). Taken together, these data strongly support the potential for a BMPS- targeting therapeutic to translate into a therapy for AD and other diseases where cholesterol dysregulation plays a role in disease.

[00287] Furthermore, atherosclerosis is driven by cholesterol deposition in porous arteries. Additionally, it has been shown that cyclodextrins can also ameliorate atherosclerotic pathology by restoring cholesterol homeostasis and trafficking (Marques et al. Front. Cell. Dev. Biol. 9:658995 (2021)). BMPS-mediated reduction of cholesterol would also then be expected to resolve this key pathology and thus a BMPS-targcting therapeutic would be expected to translate into a therapy for atherosclerosis and other diseases where cholesterol dysregulation plays a role in disease.

Example 8 Exogenous PGRN addition increases BMP synthesis in vitro

[00288] Preparation of Isolated Lysosomal Protein: In order to provide isolated lysosomal protein, one lobe of liver was dissected from a 4-month old female Grn+/- mice (Jackson lab). This tissue was then homogenized and fractionated by centrifugation (1,000 g for 2 min.) to provide 8 mL of supernatant. To this supernatant was added 500 pL of 3X-HA beads (Thermo Scientific) that had been pre washed with IxPBS three times prior to addition. This mixture was then incubated for 15 min followed by filtering of the supernatant and washing the beads three times with 10 ml IxPBS. Protein was then eluted by mixing the beads with 200 pl of ultrapure water (Thermo) and incubated at 37 °C for 30 min. Protein concentration was determined by BCA method.

[00289] Preparation ofPRGN protein: A pTwist CMV vector that encoded PGRN-6XHis was transfected to Expi293 cells at 0.5 pg/ml, transfected Expi293 cells were cultured in Expi293 Expression Medium for 3 more days followed by harvesting the supernatants (Thermo). Supernatants were then filtered with 0.22 pM filter and PGRN-6XHis were purified with Ni- NTA resin (Thermo).

[00290] Exogenous PRGN effect on BMP Production-. 10 pg of isolated lysosomal protein was then mixed with 20 pM of deuterated-PG 16:0/18:1 (D5-PG 16:0/18:1) followed by the addition of either 10 pM 6XHis tagged PGRN or IXPBS solution (Veh). A portion of the reaction mixture was collected for lipid extraction at indicated time points and measured for deuterated BMP 18:1/18:1 (D5-BMP 18:1/18:1) abundance (FIG. 24).

Example 9

Exogenous BMPS treatment rescues defective GCase activity in Grn knockout cells

[00291] Activity of GCase enzyme in BMDM derived from Grn +/- or Grn -/- mice (Jackson Lab, Strain 013175) was quantified by signal intensity of LysoFQ-GBA (Deen et al. PNAS, 2022, 119, e2200553119) after overnight treatment with either vehicle treatment (IXPBS), 50 nM BMPS-6XHis (BMPS) or 50 nM loss-of-function BMPS mutant (BMPS C231 A, labeled as mut BMPS).

[00292] As shown in FIG. 25, treatment with BMPS restored GCase activity in GRN knockout cells. GCase activity was not restored with the loss-of-function BMPS mutant, demonstrating BMPS enzymatic activity is required for rescue of GCase activity, which is as a well-known enzyme encoded by the GBA gene and the dysfunction of this gene product is thought to be causal in both monogenic and complex neurodegenerative diseases including Parkinson’s disease (PD) and frontotemporal dementia (FTD) (Valdez et al. Hum. Mol. Genet. 29(5):716-26 (2020); Arrant et al. Acta Neuropath. Commun. 7:218 (2019); Poewe et al. Nat. Rev. Dis Primers 3:17013 (2017)). In support of these data and as shown in FIG. 24, the supplementation of heterozygous loss-of-function GRN cells (GRN+/-) with recombinant functional PRGN (the gene product of GRN) enhances BMP production suggesting BMP loss is indeed a direct consequence of reduced GRN function. Importantly, GRN (otherwise known as CLN11, one of 14 genes in the family to which BMPS (CLN5) belongs) encodes the PGRN protein, and PGRN haploin sufficiency is the genetic cause of 10-15% of FTD cases (Kao et al. Nat. Rev.

Neurosci. l8:325-33 (2017)). Furthermore, biallelic loss of functions mutations to GRN (CLN11) causes NCL and results in a dramatic depletion in BMP levels, a highly comparable clinical manifestation of NCL caused by biallelic loss of function mutations to BMPS CLN5) (Kao et al. Nat. Rev. Neurosci. l8'.325-33 (2017)).

[00293] The therapeutic relevance of a BMPS therapeutic extends into other complex neurodegenerative diseases including Parkinson’s disease (PD). As demonstrated above, the activity of BMPS is required for and can stimulate GCase, the enzyme encoded by the GBA gene. The GBA gene is the most frequently mutated gene in PD with hundreds of unique mutations known to result in decreased GCase activity leading to the accumulation of the GCase substrate glucosylceramide as well as alpha-synuclein, ultimately leading to neurotoxicity and development of PD (Smith et al. Cells 11(8): 1261 (2022); Riboldi et al. Cells 8(4):364 (2019); Mazzulli et al. Cell 146(l):37-52 (2011)). Beyond GBA mutation carriers, the metabolism of glucosylceramide is defective across a spectrum of PD patients, from other genetically defined cohort to idiopathic disease (Poewe et al. Nat. Rev. Dis Primers 3:17013 (2017)).

[00294] Additionally, PD patients that carry mutations in the LRRK2 gene that lead to a hyperactive form of the kinase gene product LRRK2, where carrying such a mutation is among the strongest risk factors for developing PD (Taymans et al. npj Parkinson’ s disease 9:104 (2023); Rui ct al. Curr. Neuropharmacol. 16(9): 1348-57 (2018)). LRRK2 hyperactivity derived from these disease-causing genetic mutations results in aberrant secretion of BMP, leading to decreased BMP levels in tissue and an increase in urinary levels of PD patients (Jennings et al. Sci. Transl. Med. 14(648):eabj2658 (2022); Alcalay et al. Movement Disord. 35(1): 134-41 (2020); Gomes et al. npj Parkinson’ s disease 9:52 (2023); Maloney et al. bioRXiv (2022) doi: 10.1101/2022.12.19.521070). Beyond LRRK2 mutation carriers, levels of BMP have recently been shown to be dysregulated in other genetically defined PD cohorts as well as in idiopathic PD (Gomes et al. npj Parkinson’ s disease 9:52 (2023). Taken together, these data strongly support the potential for a BMPS therapeutic to translate into a therapy for PD, FTD, and other neurodegenerative disease as well as other diseases where lysosomal lipid metabolism and recycling is dysfunctional, including lysosomal storage diseases (e.g., NCL) and cardiovascular diseases.

Example 10 BMPS knockout cells exhibit defective GCase activity

[00295] Activity of GCase enzyme in BMDM derived from Cln5 +/- or Cln5 -I- mice (Kopra et al. Hum Mol. Genet. 13:2893-906 (2005)) was quantified by signal intensity of LysoFQ-GBA (Deen et al. PNAS, 2022, 119, e2200553119) after overnight treatment with either vehicle treatment (1XPBS), 50 nM BMPS-6XHis (BMPS) or 50 nM loss-of-function BMPS mutant (BMPS C231A, labeled as mut BMPS).

[00296] As shown in FIG. 26, BMPS knockout cells had decreased GCase activity which was rescued by treatment with BMPS, but not the loss-of-function BMPS mutant. The dependence of GCase activity on functional BMPS provides additional support for the relevance of a BMPS therapeutic in neurodegeneration including FTD and PD, as discussed above, given reduction of GCase activity and accumulation of GCase substrate glucosylceramide are a hallmark of these diseases.

Example 11 BMPS gene replacement restores BMP synthesis in BMPS KO cells

[00297] Wild-type and CLN5 KO HEK293 cells (0.6 million) were seeded on each well of a 6-well plate.CLN5 KO cells were transfected with 1 pg of the mammalian expressing vector pRK5 that expressed the CLN5-FLAG (KO + FLAG-CLN5) using PEImax (Warrington, PA). Vehicle transfected WT HEK293 cells (WT + Veh) and CLN5 KO HEK 293 cells (KO + Veh) were set as controls, where these control cells were subjected to the PEImax protocol adding ultrapure water (Invitrogen) in place of any gene vector. Whole cell lipid extraction was performed 2 days post transfection followed by measurement with QQQ mass spec using targeted methods to identify various species of endogenous lipids, including BMPs, Hemi-BMPs, PCs and PGs (for comparable method see Medoh et al. Science 2023, 381, 1182).

[00298] As shown in FIG. 27, 2-day transgene of CZJV5-FLAG rescued significantly increased synthesis of BMP. Mass spec reading of all lipids were normalized to POPC values and then normalized to each lipid of WT vehicle control.

[00299] Additionally, A AV-mediated genetic replacement of the CLN5 gene in CLN5 KO cells was explored. First, adeno-associated viral (AAV) vectors were produced via triple transfection of AAV-293 cells (Agilent Technologies, Inc., Santa Clara, CA) followed by iodixanol gradient ultracentrifugation, buffer exchange, and ultrafiltration. Genomic titers were calculated via digital quantitative PCR (QIAcuity One, QIAGEN, Redwood City, CA) using primers/probes targeted to WPRE and ITR sequences. To assess the effect of AAV-mediated gene replacement, 1 million WT HEK293T or CLN5 KO HEK293T cells were seeded in each well of a poly-L-lysine coated 6-well plate. In the treatment group, AAV expressing FLAG- CLN5 was added during seeding at 10E10 virus particles/mL to CLN5 KO cells. For control groups, 1 pl of PBS were added to each well containing either WT HEK293T or CLN5 KO HEK293T cells. Cells were incubated for 24 hours before collecting cells for lipidomic analysis of BMP or incubated for 48 hours before collecting cells for Western blot analysis. Reagents used for Western blot analysis included anti-FLAG antibody (Cell signaling technology, 14793), anti-CLN5 antibody (Abeam 170899), LAMP1 antibody (Cell signaling technology, 9091), and GAPDH antibody (Cell signaling technology, 5174). Abundance of BMP was measured with triple quadrupole mass spectrometry.

[00300] FIG. 28 shows characterization and validation of AAV-mediated gene replacement of the BMPS (CLN5) gene in CLN5 knock-out cells with western blot analysis of BMPS protein levels in wildtype HEK293T cells (WT), CLN5 knockout HEK293T cells (KO), and CLN5 knockout HEK293T cells treated with an AAV vector containing the gene for FLAG-tagged BMPS (KO+AAV). FIG. 29 shows BMPS gene replacement after viral gene replacement restores BMP synthesis in BMPS knockout cells. Abundance of BMPS expression was measured with triple quadrupole mass spectrometry in wild-type (WT) and BMPS knockout HEK293T cells (CLN5 labeled “KO”) with vehicle and BMPS knockout HEK293T cells treated with a FLAG-tagged BMPS AAV vector (KO + AAV-FLAG-CLN5). Taken together, these data show that treatment with AAV encoding the gene for BMPS (CLN5) was able to successfully restore expression of BMPS protein on a knockout background, providing restored expression of the gene product BMPS in fully functional form rescuing the loss of BMP observed after initial knockout and suggesting that a gene therapy or other mechanism for replacement of alterative of the BMPS gene may prove to be a consequential therapeutic for diseases with dysregulated BMP levels or improper lysosomal function including but not limited to LSDs and neurodegenerative diseases.

[00301] BMPS Sequence (SEQ ID NO: 1) MAQEVDTAQGAEMRRGAGAARGRASWCWALALLWLAVVPGWSRVSGIPSRRHWPVP YKRFDFRPKPDPYCQAKYTFCPTGSPIPVMEGDDDIEVFRLQAPVWEFKYGDLLGHLKI MHDAIGFRSTLTGKNYTMEWYELFQLGNCTFPHLRPEMDAPFWCNQGAACFFEGIDDV HWKENGTLVQVATISGNMFNQMAKWVKQDNETGIYYETWNVKASPEKGAETWFDSY DCSKFVLRTFNKLAEFGAEFKNIETNYTRIFLYSGEPTYLGNETSVFGPTGNKTLGLAIKR FYYPFKPHLPTKEFLLSLLQIFDAVIVHKQFYLFYNFEYWFLPMKFPFIKITYEEIPLPIRNK TESGE

[00302] BMPS Sequence Isoform CRA_a (SEQ ID NO:2) MRRNLRLGPSSGADAQGQGAPRPGLAAPRMLLPPASQASRGSGSTGCSLMAQEVDTAQ GAEMRRGAGAARGRASWCWALALLWLAVVPGWSRVSGIPSRRHWPVPYKRFDFRPKP DPYCQAKYTFCPTGSPIPVMEGDDDIEVFRLQAPVWEFKYGDLLGHLKIMHDAIGFRST LTGKNYTMEWYELFQLGNCTFPHLRPEMDAPFWCNQGAACFFEGIDDVHWKENGTLV QVATISGNMFNQMAKWVKQDNETGIYYETWNVKASPEKGAETWFDSYDCSKFVLRTF NKLAEFGAEFKNIETNYTRIFLYSGEPTYLGNETSVFGPTGNKTLGLAIKRFYYPFKPHLP TKEFLLSLLQIFDAVIVHKQFYLFYNFEYWFLPMKFPFIKITYEEIPLPIRNKTLSGL [00303] BMPS-6xHis WT (SEQ ID NOG) TCCCAGGTCCAACTGCACCTCGGTTCTATCGATTGAATTCACCATGGCCCAGGAGGT CGACACAGCACAGGGAGCAGAGATGAGACGGGGCGCCGGGGCCGCCAGAGGACGG GCCTCCTGGTGTTGGGCACTCGCCCTCCTGTGGCTGGCTGTCGTGCCTGGGTGGAGC AGAGTTTCAGGCATCCCATCTCGCCGACACTGGCCAGTTCCTTATAAGAGATTTGAC

TTCAGACCAAAGCCCGACCCGTATTGCCAGGCCAAGTACACATTTTGCCCTACTGGG

TCACCCATACCCGTCATGGAGGGAGACGATGACATCGAAGTGTTTAGATTGCAAGC

CCCCGTGTGGGAGTTCAAGTACGGTGACCTGCTTGGCCATCTGAAAATCATGCATGA

CGCAATAGGTTTCCGGTCTACCCTCACAGGAAAGAATTACACCATGGAGTGGTATGA

ACTCTTCCAGCTGGGCAATTGCACATTCCCACATCTGAGACCTGAGATGGATGCACC

ATTCTGGTGTAACCAGGGCGCTGCCTGCTTCTTCGAAGGGATCGACGACGTGCATTG

GAAAGAGAATGGCACCCTGGTACAGGTCGCGACAATTAGCGGCAATATGTTTAACC

AGATGGCCAAATGGGTGAAGCAGGACAACGAGACAGGCATATACTACGAAACATG

GAACGTGAAGGCATCACCTGAGAAGGGCGCTGAAACTTGGTTCGACAGTTATGATT

GCAGCAAATTTGTCCTGAGAACTTTTAATAAGCTGGCAGAGTTCGGAGCCGAATTCA

AAAATATTGAAACCAATTACACCAGGATCTTTCTTTATTCCGGCGAGCCCACTTACC

TGGGCAACGAAACCTCAGTGTTCGGTCCTACCGGGAATAAGACGCTCGGTTTGGCCA

TCAAACGCTTCTACTACCCTTTCAAGCCACATTTGCCCACCAAGGAGTTCCTCCTGA

GTCTCCTGCAGATTTTTGATGCTGTGATCGTCCATAAGCAGTTTTACTTGTTTTACAA

TTTCGAATATTGGTTCCTCCCCATGAAATTCCCTTTCATCAAGATCACATACGAGGA

GATCCCCCTCCCCATTAGGAACAAGACTCTGTCAGGCCTCGGGGGCGGCGGAAGCC

ACCATCACCACCATCACTAAGCGGCCGCTAAGTAAGTAAGGATCCCCAGCTTGGCC

GCCA

[00304] C231S BMPS-6xHis (SEQ ID NO:4)

TCCCAGGTCCAACTGCACCTCGGTTCTATCGATTGAATTCACCATGGCCCAGGAGGT

CGACACAGCACAGGGAGCAGAGATGAGACGGGGCGCCGGGGCCGCCAGAGGACGG

GCCTCCTGGTGTTGGGCACTCGCCCTCCTGTGGCTGGCTGTCGTGCCTGGGTGGAGC

AGAGTTTCAGGCATCCCATCTCGCCGACACTGGCCAGTTCCTTATAAGAGATTTGAC

TTCAGACCAAAGCCCGACCCGTATTGCCAGGCCAAGTACACATTTTGCCCTACTGGG

TCACCCATACCCGTCATGGAGGGAGACGATGACATCGAAGTGTTTAGATTGCAAGC

CCCCGTGTGGGAGTTCAAGTACGGTGACCTGCTTGGCCATCTGAAAATCATGCATGA

CGCAATAGGTTTCCGGTCTACCCTCACAGGAAAGAATTACACCATGGAGTGGTATGA

ACTCTTCCAGCTGGGCAATTGCACATTCCCACATCTGAGACCTGAGATGGATGCACC

ATTCTGGTGTAACCAGGGCGCTGCCTGCTTCTTCGAAGGGATCGACGACGTGCATTG

GAAAGAGAATGGCACCCTGGTACAGGTCGCGACAATTAGCGGCAATATGTTTAACC AGATGGCCAAATGGGTGAAGCAGGACAACGAGACAGGCATATACTACGAAACATG

GAACGTGAAGGCATCACCTGAGAAGGGCGCTGAAACTTGGTTCGACAGTTATGATA

GTAGCAAATTTGTCCTGAGAACTTTTAATAAGCTGGCAGAGTTCGGAGCCGAATTCA

AAAATATTGAAACCAATTACACCAGGATCTTTCTTTATTCCGGCGAGCCCACTTACC

TGGGCAACGAAACCTCAGTGTTCGGTCCTACCGGGAATAAGACGCTCGGTTTGGCCA

TCAAACGCTTCTACTACCCTTTCAAGCCACATTTGCCCACCAAGGAGTTCCTCCTGA

GTCTCCTGCAGATTTTTGATGCTGTGATCGTCCATAAGCAGTTTTACTTGTTTTACAA

TTTCGAATATTGGTTCCTCCCCATGAAATTCCCTTTCATCAAGATCACATACGAGGA

GATCCCCCTCCCCATTAGGAACAAGACTCTGTCAGGCCTCGGGGGCGGCGGAAGCC

ACCATCACCACCATCACTAAGCGGCCGCTAAGTAAGTAAGGATCCCCAGCTTGGCC

GCCA

[00305] BMPS-6xHis K319E (SEQ ID NO:5)

TCCCAGGTCCAACTGCACCTCGGTTCTATCGATTGAATTCACCATGGCCCAGGAGGT

CGACACAGCACAGGGAGCAGAGATGAGACGGGGCGCCGGGGCCGCCAGAGGACGG

GCCTCCTGGTGTTGGGCACTCGCCCTCCTGTGGCTGGCTGTCGTGCCTGGGTGGAGC

AGAGTTTCAGGCATCCCATCTCGCCGACACTGGCCAGTTCCTTATAAGAGATTTGAC

TTCAGACCAAAGCCCGACCCGTATTGCCAGGCCAAGTACACATTTTGCCCTACTGGG

TCACCCATACCCGTCATGGAGGGAGACGATGACATCGAAGTGTTTAGATTGCAAGC

CCCCGTGTGGGAGTTCAAGTACGGTGACCTGCTTGGCCATCTGAAAATCATGCATGA

CGCAATAGGTTTCCGGTCTACCCTCACAGGAAAGAATTACACCATGGAGTGGTATGA

ACTCTTCCAGCTGGGCAATTGCACATTCCCACATCTGAGACCTGAGATGGATGCACC

ATTCTGGTGTAACCAGGGCGCTGCCTGCTTCTTCGAAGGGATCGACGACGTGCATTG

GAAAGAGAATGGCACCCTGGTACAGGTCGCGACAATTAGCGGCAATATGTTTAACC

AGATGGCCAAATGGGTGAAGCAGGACAACGAGACAGGCATATACTACGAAACATG

GAACGTGAAGGCATCACCTGAGAAGGGCGCTGAAACTTGGTTCGACAGTTATGATT

GCAGCAAATTTGTCCTGAGAACTTTTAATAAGCTGGCAGAGTTCGGAGCCGAATTCA

AAAATATTGAAACCAATTACACCAGGATCTTTCTTTATTCCGGCGAGCCCACTTACC

TGGGCAACGAAACCTCAGTGTTCGGTCCTACCGGGAATAAGACGCTCGGTTTGGCCA

TCAAACGCTTCTACTACCCTTTCAAGCCACATTTGCCCACCAAGGAGTTCCTCCTGA

GTCTCCTGCAGATTTTTGATGCTGTGATCGTCCATGAACAGTTTTACTTGTTTTACAA

TTTCGAATATTGGTTCCTCCCCATGAAATTCCCTTTCATCAAGATCACATACGAGGA GATCCCCCTCCCCATTAGGAACAAGACTCTGTCAGGCCTCGGGGGCGGCGGAAGCC

ACCATCACCACCATCACTAAGCGGCCGCTAAGTAAGTAAGGATCCCCAGCTTGGCC GCCA

[00306] BMPS-6xHis HK318/9EE (SEQ ID NO:6)

TCCCAGGTCCAACTGCACCTCGGTTCTATCGATTGAATTCACCATGGCCCAGGAGGT

CGACACAGCACAGGGAGCAGAGATGAGACGGGGCGCCGGGGCCGCCAGAGGACGG

GCCTCCTGGTGTTGGGCACTCGCCCTCCTGTGGCTGGCTGTCGTGCCTGGGTGGAGC

AGAGTTTCAGGCATCCCATCTCGCCGACACTGGCCAGTTCCTTATAAGAGATTTGAC

TTCAGACCAAAGCCCGACCCGTATTGCCAGGCCAAGTACACATTTTGCCCTACTGGG

TCACCCATACCCGTCATGGAGGGAGACGATGACATCGAAGTGTTTAGATTGCAAGC

CCCCGTGTGGGAGTTCAAGTACGGTGACCTGCTTGGCCATCTGAAAATCATGCATGA

CGCAATAGGTTTCCGGTCTACCCTCACAGGAAAGAATTACACCATGGAGTGGTATGA

ACTCTTCCAGCTGGGCAATTGCACATTCCCACATCTGAGACCTGAGATGGATGCACC

ATTCTGGTGTAACCAGGGCGCTGCCTGCTTCTTCGAAGGGATCGACGACGTGCATTG

GAAAGAGAATGGCACCCTGGTACAGGTCGCGACAATTAGCGGCAATATGTTTAACC

AGATGGCCAAATGGGTGAAGCAGGACAACGAGACAGGCATATACTACGAAACATG

GAACGTGAAGGCATCACCTGAGAAGGGCGCTGAAACTTGGTTCGACAGTTATGATT

GCAGCAAATTTGTCCTGAGAACTTTTAATAAGCTGGCAGAGTTCGGAGCCGAATTCA

AAAATATTGAAACCAATTACACCAGGATCTTTCTTTATTCCGGCGAGCCCACTTACC

TGGGCAACGAAACCTCAGTGTTCGGTCCTACCGGGAATAAGACGCTCGGTTTGGCCA

TCAAACGCTTCTACTACCCTTTCAAGCCACATTTGCCCACCAAGGAGTTCCTCCTGA

GTCTCCTGCAGATTTTTGATGCTGTGATCGTCGAAGAACAGTTTTACTTGTTTTACAA

TTTCGAATATTGGTTCCTCCCCATGAAATTCCCTTTCATCAAGATCACATACGAGGA

GATCCCCCTCCCCATTAGGAACAAGACTCTGTCAGGCCTCGGGGGCGGCGGAAGCC

ACCATCACCACCATCACTAAGCGGCCGCTAAGTAAGTAAGGATCCCCAGCTTGGCC GCCA

[00307] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. [00308] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[00309] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIMS:

1. A method of stimulating and/or maintaining lysosomal function in a subject in need thereof, the method comprising increasing the level and/or the activity bis(monoacylglycero)phosphate (BMP) synthase in the subject by administering to the subject an effective amount of: a BMP synthase activator or a composition thereof; and/or a BMP synthase, a catalytically active fragment or variant thereof, or a nucleic acid encoding BMP synthase or a catalytically active fragment or variant thereof.

2. The method of claim 1, wherein the activator comprises a small molecule activator of BMP synthase.

3. The method of claim 1 or 2, wherein the activator comprises progranulin, granulin peptides, or derivatives thereof.

4. The method of any of claims 1-3, wherein the activator comprises phosphatidylglycerol or derivatives thereof, lysophosphatidylglycerol or derivatives thereof, or combinations thereof.

5. The method of any of claims 1-4, wherein the activator comprises a protease inhibitor.

6. The method of any of claims 1-5, wherein the BMP synthase comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 1 or 2.

7. The method of any of claims 1-6, wherein the BMP synthase comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 1 or 2.

8. The method of any of claims 1-7, wherein the BMP synthase comprises an amino acid sequence of SEQ ID NO: 1 or 2.

9. The method of any of claims 1-8, wherein the BMP synthase activator or BMP synthase or catalytically active fragment thereof is linked to a polypeptide configured to bind to a bloodbrain barrier (BBB) receptor.

10. The method of claim 9, wherein the polypeptide comprises a receptor-binding domain from an apolipoprotein.

11. The method of any of claims 1-10, wherein the BMP synthase activator or BMP synthase or catalytically active fragment thereof is linked to a polypeptide comprising a first Fc region.

12. The method of claim 11 , wherein the first Fc region is derived from an immunoglobulin IgG Fc region.

13. The method of claim 11 or 12, wherein the polypeptide is an antibody or fragment thereof.

14. The method of claim 13, wherein the antibody or fragment thereof is configured to bind to a blood-brain barrier (BBB) receptor.

15. The method of any of claims 11-14, further comprising administering a polypeptide comprising a second Fc region configured to form a dimer with the first Fc region.

16. The method of any of claims 1-15, further comprising activating autophagy or autophagy related pathways, increasing the level and/or the activity of transcription factor EB (TFEB), or a combination thereof.

17. The method of any of claims 1-16, further comprising administering at least one immune modulator or neuroprotective compound.

18. The method of any of claims 1-17, wherein the subject has or is suspected of having a disease or disorder characterized by lysosomal dysfunction, decreased lipid catabolism, and/or increased cholesterol accumulation.

19. The method of any of claims 1-18, wherein the subject has or is suspected of having a neurodegenerative disease.

20. The method of claim 19, wherein the neurodegenerative disease is selected from Niemann- Pick disease, frontotemporal dementia (FTD), Parkinson’s disease, Alzheimer’s disease, a neuronal ceroid lipofuscinosis, amyotrophic lateral sclerosis (ALS), and Dementia with Lewy Bodies (DLB).

21. The method of claim 20, wherein the neuronal ceroid lipofuscinosis is selected from neuronal ceroid lipofuscinosis type 1 (CLN1), neuronal ceroid lipofuscinosis type 2 (CLN2), neuronal ceroid lipofuscinosis type 3 (CLN3), neuronal ceroid lipofuscinosis type 4 (CLN4), neuronal ceroid lipofuscinosis type 5 (CLN5), neuronal ceroid lipofuscinosis type 6 (CLN6), neuronal ceroid lipofuscinosis type 7 (CLN7), neuronal ceroid lipofuscinosis type 8 (CLN8), neuronal ceroid lipofuscinosis type 9 (CLN9), neuronal ceroid lipofuscinosis type 10 (CLN10), neuronal ceroid lipofuscinosis type 11 (CLN11), neuronal ceroid lipofuscinosis type 12 (CLN12), neuronal ceroid lipofuscinosis type 13 (CLN13), and neuronal ceroid lipofuscinosis type 14 (CLN14).

22. The method of any of claims 1-21, wherein the subject has or is suspected of having a lysosomal storage disorder.

23. The method of claim 22, wherein the lysosomal storage disorder is selected from: Gaucher’s disease, metachromatic leukodystrophy, GM1 gangliosidosis, Tay-Sachs disease, Sandhoff disease, Fabry disease, Farber disease, Wolman disease, mucolipidosis Types I, II/III and IV, Type I mucopolysaccharidoses, Type II mucopolysaccharidoses, Type III mucopolysaccharidoses. Type IV mucopolysaccharidoses, Type VI mucopolysaccharidoses, and Krabbe disease.

24. The method of any of claims 1-23, wherein the subject has or is suspected of having drug- induced phospholipidosis, familial hypercholesterolemia (FH), a cardiovascular disease, atherosclerosis, obesity, a fatty liver disease, or any combination thereof.

25. The method of any of claims 1-24, wherein the administration is via the cerebrospinal fluid.

26. The method of any of claims 1-25, wherein the administration is intrathecal, intracistemal, or intracerebroventricular.

27. The method of any of claims 1-26, wherein the administration comprises systemic administration.

28. A method of treating or preventing a disease or disorder in a subject comprising administering to the subject a therapeutically or prophylactically effective amount of a BMP synthase inhibitor or a composition thereof to the subject.

29. The method of claim 28, wherein the disease or disorder is selected from a Von Hippel- Lindau (VHL) related cancer, K-Ras driven cancers, lung cancer, pancreatic cancer, prostate cancer, breast cancer, cancers related to low levels of HSP70, or an infectious disease.

30. The method of claim 28 or 29, wherein the inhibitor is selected from the group consisting of a protein configured to bind BMP synthase or a substrate thereof, a gene silencing oligonucleotide, a small molecule inhibitor of BMP synthase, a negative allosteric modulating agent, a protease, and combinations thereof.

31 . The method of any of claims 28-30, wherein the inhibitor comprises a dominant negative BMPS or variant or fragment thereof.

32. The method of claim 31, wherein the dominant negative BMPS or variant or fragment thereof comprises one or more mutations in the catalytic active site.

33. The method of claim 31 or 32, wherein the dominant negative BMPS or variant or fragment thereof comprises one or more mutations in cysteine 231, histidine 117, & glutamate 134 in reference to SEQ ID NO: 1.

34. The method of any of claims 28-33, wherein the inhibitor comprises a negative allosteric modulating agent.

35. The method of claim 34, wherein the negative allosteric modulating agent is configured to bind to a binding site of BMP synthase, a catalytically active fragment or variant thereof, comprising one, two or all residues at positions 73, 143, and 296 in reference to SEQ ID NO: 1.

36. The method of claim 35, wherein the binding site comprises one, two or all of K73, N143, and K296, in reference to SEQ ID NO: 1.

37. The method of any of claims 28-36, wherein the BMP synthase inhibitor comprises a glycerophosphodiester.

38. The method of any of claims 28-37, wherein the inhibitor is selected from the group consisting of glycerophosphatidylserine (GPS), glycerophosphatidylethanolamine (GPE), glycerophosphoglycerol (GPG), glycosylphosphatidylinositol (GPI), glycerophosphatidylcholine (GPC), and combinations thereof.

39. A method of treating or preventing a disease or disorder in a subject, comprising administering to the subject an effective amount of BMP synthase, a catalytically active fragment or variant thereof, or a nucleic acid encoding BMP synthase, a catalytically active fragment or variant thereof to the subject.

40. The method of claim 39, wherein the disease or disorder is characterized by lysosomal dysfunction, decreased lipid catabolism, and/or increased cholesterol accumulation.

41. The method of claim 39 or 40, wherein the disease or disorder is a neurodegenerative disease.

42. The method of claim 41 , wherein the neurodegenerative disease is selected from Niemann- Pick disease, frontotemporal dementia (FTD), Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), and Dementia with Lewy Bodies (DLB).

43. The method of claim 41, wherein the neurodegenerative disease is selected from neuronal ceroid lipofuscinosis type 1 (CLN1), neuronal ceroid lipofuscinosis type 2 (CLN2), neuronal ceroid lipofuscinosis type 3 (CLN3), neuronal ceroid lipofuscinosis type 4 (CLN4), neuronal ceroid lipofuscinosis type 6 (CLN6), neuronal ceroid lipofuscinosis type 7 (CLN7), neuronal ceroid lipofuscinosis type 8 (CLN8), neuronal ceroid lipofuscinosis type 9 (CLN9), neuronal ceroid lipofuscinosis type 10 (CLN10), neuronal ceroid lipofuscinosis type 11 (CLN11), neuronal ceroid lipofuscinosis type 12 (CLN12), neuronal ceroid lipofuscinosis type 13 (CLN13), and neuronal ceroid lipofuscinosis type 14 (CLN14).

44. The method of any of claims 39-43, wherein the subject has or is suspected of having a lysosomal storage disorder.

45. The method of claim 44, wherein the lysosomal storage disorder is selected from: Gaucher’s disease, metachromatic leukodystrophy, GM1 gangliosidosis, Tay-Sachs disease, Sandhoff disease, Fabry disease, Farber disease, Wolman disease, mucolipidosis Types I, II/III and IV, Type I mucopolysaccharidoses, Type II mucopolysaccharidoses, Type III mucopolysaccharidoses, Type IV mucopolysaccharidoses, Type VI mucopolysaccharidoses, and Krabbe disease.

46. The method of any of claims 39-45, wherein the subject has or is suspected of having drug- induced phospholipidosis, familial hypercholesterolemia (FH), a cardiovascular disease, atherosclerosis, obesity, a fatty liver disease, or any combination thereof.

47. The method of any of claims 39-46, wherein the BMP synthase comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 1 or 2.

48. The method of any of claims 39-47, wherein the BMP synthase comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 1 or 2.

49. The method of any of claims 39-48, wherein the BMP synthase or catalytically active fragment thereof is linked to a polypeptide configured to bind to a blood-brain barrier (BBB) receptor.

50. The method of claim 49, wherein the polypeptide comprises a receptor-binding domain from an apolipoprotein.

51. The method of any of claims 39-50, wherein the BMP synthase or catalytically active fragment thereof is linked to a polypeptide comprising a first Fc region.

52. The method of claim 51, wherein the first Fc region is derived from an immunoglobulin IgG Fc region.

53. The method of claim 51 or 52, wherein the polypeptide is an antibody or fragment thereof.

54. The method of claim 53, wherein the antibody or fragment thereof is configured to bind to a blood-brain barrier (BBB) receptor.

55. The method of any of claims 51-54, further comprising administering a polypeptide comprising a second Fc region configured to form a dimer with the first Fc region.

56. The method of any of claims 39-55, wherein the administration is via the cerebrospinal fluid.

57. The method of any of claims 39-56, wherein the administration is intrathecal, intracistemal, or intracerebro ventricular.

58. The method of any of claims 39-55, wherein the administration comprises systemic administration.

59. A nucleic acid comprising an engineered BMP synthase, a catalytically active fragment or variant thereof, a fusion protein comprising a BMP synthase and an Fc domain, or a dominant negative variant BMP synthase.

60. A vector comprising the nucleic acid of claim 59.

61. A cell or population of cells comprising the nucleic acid of claim 59 or the vector of claim

60.

62. The cell or population of cells of claim 61 , wherein the cell or population of cells are in vitro or ex vivo.

63. A BMP synthase activator or a composition thereof; and/or a BMP synthase, a catalytically active fragment or variant thereof, or a nucleic acid encoding BMP synthase or a catalytically active fragment or variant thereof for use in stimulating and/or maintaining lysosomal function.

64. A BMP synthase activator or a composition thereof; and/or a BMP synthase, a catalytically active fragment or variant thereof, or a nucleic acid encoding BMP synthase or a catalytically active fragment or variant thereof for use in treating a disease or disorder characterized by lysosomal dysfunction, decreased lipid catabolism, and/or increased cholesterol accumulation.

65. A BMP synthase inhibitor or a composition thereof for treating or preventing a disease or disorder in a subject comprising administering to the subject a therapeutically or prophylactically effective amount of a BMP synthase inhibitor or a composition thereof to the subject, wherein the disease or disorder is selected from a Von Hippel-Lindau (VHL) related cancer, K-Ras driven cancers, lung cancer, pancreatic cancer, prostate cancer, breast cancer, cancers related to low levels of HSP70, or an infectious disease.

66. A compound configured to bind to a binding site of BMP synthase, a catalytically active fragment or variant thereof, comprising one, two or all residues at positions 73, 143, and 296, in reference to SEQ ID NO: 1.

67. The compound of claim 66, wherein the compound comprises one, two or all K73, N143, and K296, in reference to SEQ ID NO: 1.

68. A method of inhibiting BMP synthesis comprising binding a compound of claim 66 or 67 to BMP synthase, a catalytically active fragment or variant thereof.

69. A method of inhibiting BMP synthesis comprising binding a glycerophosphodiester to BMP synthase, a catalytically active fragment or variant thereof.