Fabry Disease Mouse Research Articles

Usefulness of antibody-drug conjugate as preconditioning for hematopoietic stem cell-targeted gene therapy in wild-type and Fabry disease mouse models

BackgroundFabry disease (FD) is characterized by deficient activity of α-galactosidase A (GLA). Consequently, globotriaosylceramide (Gb3) accumulates in various organs, causing cardiac, renal, and cerebrovascular damage. Gene therapies for FD have been investigated in humans. Strong conditioning is required for hematopoietic stem cell-targeted gene therapy (HSC-GT). However, strong conditioning leads to various side effects and should be avoided. In this study, we tested antibody-based conditioning for HSC-GT in wild-type and FD model mice. MethodsAfter preconditioning with an antibody-drug conjugate, HSC-GT using a lentiviral vector was performed in wild-type and Fabry model mice. In the wild-type experiment, the EGFP gene was introduced into HSCs and transplanted into preconditioned mice, and donor chimerism and EGFP expression were analyzed. In the FD mouse model, the GLA gene was introduced into HSCs and transplanted into preconditioned Fabry mice. GLA activity and Gb3 accumulation in the organs were analyzed. ResultsIn the wild-type mouse experiment, when anti-CD45 antibody-drug conjugate was used, the percentage of donor cells at 6 months was 64.5%, and 69.6% of engrafted donor peripheral blood expressed EGFP. When anti-CD117 antibody-drug conjugate and ATG were used, the percentage of donor cells at 6 months was 80.7%, and 73.4% of engrafted donor peripheral blood expressed EGFP. Although large variations in GLA activity among mice were observed in the FD mouse experiment for both preconditioning regimens, Gb3 was significantly reduced in many organs. ConclusionsAntibody-based preconditioning may be an alternative preconditioning strategy for HSC-GT for treating FD.

Evaluating the Metabolic Basis of α-Gal A mRNA Therapy for Fabry Disease.

mRNA injection-based protein supplementation has emerged as a feasible treatment for Fabry disease. However, whether the introduction of LNP-encapsulated mRNA results in the alteration of metabolomics in an in vivo system remains largely unknown. In the present study, α-galactosidase A (α-Gal A) mRNA was generated and injected into the Fabry disease mouse model. The α-Gal A protein was successfully expressed. The level of globotriaosylsphingosine (Lyso-Gb3), a biomarker for Fabry disease, as well as pro-inflammatory cytokines such as nuclear factor kappa-B (NF-κB), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α), were greatly decreased compared to the untreated control, indicating the therapeutic outcome of the mRNA drug. Metabolomics analysis found that the level of 20 metabolites was significantly altered in the plasma of mRNA-injected mice. These compounds are primarily enriched in the arachidonic acid metabolism, alanine, aspartate and glutamate metabolism, and glycolysis/gluconeogenesis pathways. Arachidonic acid and 5-hydroxyeicosatetraenoic acid (5-HETE), both of which are important components in the eicosanoid pathway and related to inflammation response, were significantly increased in the injected mice, possibly due to the presence of lipid nanoparticles. Moreover, mRNA can effectively alter the level of metabolites in the amino acid and energy metabolic pathways that are commonly found to be suppressed in Fabry disease. Taken together, the present study demonstrated that in addition to supplementing the deficient α-Gal A protein, the mRNA-based therapeutic agent can also affect levels of metabolites that may help in the recovery of metabolic homeostasis in the full body system.

WARBURG EFFECT CHARACTERIZES THE SKELETAL MUSCLE IN FABRY DISEASE: EXPERIMENTAL EVIDENCE AND CLINICAL IMPLICATIONS

Objective: Skeletal muscle (SM) pain and fatigue are common in Fabry disease (FD), and strongly impact the patient's quality of life. Still, the SM in FD is poorly investigated. Although energetic alterations are reported in cells from FD patients, they have never been related to fatigue and pain. Given the pivotal relevance of energetics for SM health, we undertook the investigation of the SM. Design and method: We consistently observed a reduced tolerance to aerobic activity and lactate accumulation in FD-humanized mouse model and patients. Accordingly, in sedentary mouse FD SM we detected an increase in fast/glycolytic-fibers, mirrored by an upregulation of glycolytic enzymes and glucose-transporters Results: In fibroblasts derived from FD patients, we confirmed a high glycolytic-rate and accordingly, metabolomic/lipidomic-analysis revealed that lipids are underutilized as energetic fuel in FD-patients. In the quest for a tentative mechanism, we explored analogies with genetic myopathies, that show altered expression of energetic metabolism. Specifically, we explored HIF-1 upregulation and found it increased in FD mice and patients. Consistent with our previous screening of miRNAs associated with metabolic stress in FD, we observed a significant upregulation of miR-17 in FD patients and mice. Of note, miR-17 has been associated with cellular metabolic remodeling and HIF-1 up-regulation in different contexts. In our experimental setting, a specific antagomir targeting miR-17 was able to inhibit HIF-1 accumulation and revert metabolic-remodeling in FD-cells. Conclusions: Our findings unveil an anaerobic glycolytic-switch under normoxia, known as Warburg Effect, which is induced by miR-17-mediated-upregulation of HIF-1. The miR-17/HIF-1 pathway can be a new therapeutic target in FD, and Exercise-testing and blood lactate may become a new diagnostic and monitoring-tool in FD.

Age-related neuroimmune signatures in dorsal root ganglia of a Fabry disease mouse model

Pain in Fabry disease (FD) is generally accepted to result from neuronal damage in the peripheral nervous system as a consequence of excess lipid storage caused by alpha-galactosidase A (α-Gal A) deficiency. Signatures of pain arising from nerve injuries are generally associated with changes of number, location and phenotypes of immune cells within dorsal root ganglia (DRG). However, the neuroimmune processes in the DRG linked to accumulating glycosphingolipids in Fabry disease are insufficiently understood.Therefore, using indirect immune fluorescence microscopy, transmigration assays and FACS together with transcriptomic signatures associated with immune processes, we assessed age-dependent neuroimmune alterations in DRG obtained from mice with a global depletion of α-Gal A as a valid mouse model for FD. Macrophage numbers in the DRG of FD mice were unaltered, and BV-2 cells as a model for monocytic cells did not show augmented migratory reactions to glycosphingolipids exposure suggesting that these do not act as chemoattractants in FD. However, we found pronounced alterations of lysosomal signatures in sensory neurons and of macrophage morphology and phenotypes in FD DRG. Macrophages exhibited reduced morphological complexity indicated by a smaller number of ramifications and more rounded shape, which were age dependent and indicative of premature monocytic aging together with upregulated expression of markers CD68 and CD163.In our FD mouse model, the observed phenotypic changes in myeloid cell populations of the DRG suggest enhanced phagocytic and unaltered proliferative capacity of macrophages as compared to wildtype control mice. We suggest that macrophages may participate in FD pathogenesis and targeting macrophages at an early stage of FD may offer new treatment options other than enzyme replacement therapy.

Fasudil alleviates the vascular endothelial dysfunction and several phenotypes of Fabry disease

Fabry disease (FD), a lysosomal storage disorder, is caused by defective α-galactosidase (GLA) activity, which results in the accumulation of globotriaosylceramide (Gb3) in endothelial cells and leads to life-threatening complications such as left ventricular hypertrophy (LVH), renal failure, and stroke. Enzyme replacement therapy (ERT) results in Gb3 clearance; however, because of a short half-life in the body and the high immunogenicity of FD patients, ERT has a limited therapeutic effect, particularly in patients with late-onset disease or progressive complications. Because vascular endothelial cells (VECs) derived from FD-induced pluripotent stem cells display increased thrombospondin-1 (TSP1) expression and enhanced SMAD2 signaling, we screened for chemical compounds that could downregulate TSP1 and SMAD2 signaling. Fasudil reduced the levels of p-SMAD2 and TSP1 in FD-VECs and increased the expression of angiogenic factors. Furthermore, fasudil downregulated the endothelial-to-mesenchymal transition (EndMT) and mitochondrial function of FD-VECs. Oral administration of fasudil to FD mice alleviated several FD phenotypes, including LVH, renal fibrosis, anhidrosis, and heat insensitivity. Our findings demonstrate that fasudil is a novel candidate for FD therapy.

Characterization of the G3Stg/KO Fabry disease mouse model pathology to improve preclinical to clinical translation

Characterization of the G3Stg/KO Fabry disease mouse model pathology to improve preclinical to clinical translation

Efficacy of AAV5-GLA gene therapy in manifest Fabry disease mice

Efficacy of AAV5-GLA gene therapy in manifest Fabry disease mice

Distinctive accumulation of globotriaosylceramide and globotriaosylsphingosine in a mouse model of classic Fabry disease

Fabry disease (FD) is an inherited disease caused by deficient α-galactosidase A activity that is characterized by the accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). Although plasma lyso-Gb3 is a sensitive biomarker of FD, the correlation between its concentration and clinical symptoms remains unclear. To clarify the influence of plasma Gb3 and lyso-Gb3 in a symptomatic GlatmTg(CAG-A4GALT) FD mouse model, the total contents of Gb3, lyso-Gb3 and their analogs in various organs and plasma were determined in mice with early- (5-week-old) and late-stage (20-week-old) renal dysfunction. A marked increase in total Gb3 content in the heart, kidneys, spleen, liver, small intestine, lungs, brain, and plasma was observed in the 20-week-old mice compared to that in 5-week-old mice. In contrast, the increase in lyso-Gb3 was relatively small, and the total content in the lungs and plasma was unchanged. Lyso-Gb3 analogs {lyso-Gb3(−2) and lyso-Gb3(+18)} and Gb3 analogs {Gb3(−2) and Gb3(+18)} were observed in all organs and plasma at both ages, and the percentages of the analogs were unique to specific organs. The pattern of 37 Gb3 analogs/isoforms of liver Gb3 corresponded well with that of plasma Gb3. Although the analog pattern of plasma lyso-Gb3 did not resemble that of any organ lyso-Gb3, the relative content {lyso-Gb3: lyso-Gb3(−2)} in the sum of all organs corresponded well to that of the plasma at both ages. These data indicate that liver Gb3 may contribute to the plasma Gb3 level, while plasma lyso-Gb3 may be released from all organs, and the capacity of the plasma lyso-Gb3 pool may reach a maximum at an early stage of renal dysfunction.

PS-B05-6: FABRY DISEASE MOUSE IS RESISTANT TO HIGH-SALT DIET-INDUCED HYPERTENSION PROBABLY VIA DYSFUNCTIONAL SODIUM TRANSPORTS AND AQUAPORIN 2

Background: Fabry disease is a rare X-linked lysosomal storage disorder resulting from an error in glycosphingolipid metabolism caused by the GLA gene mutation. Patients with Fabry disease often have near normal or even low systemic blood pressure although the mechanism underlying such observed finding remains unexplained. This study examined whether a high salt intake could affect transport of sodium and water and induce hypertension in mice with Fabry disease. Methods: Fabry disease model mice (B6;129-Glatm1Kul/J) and wild-type (WT) mice received 8% or normal NaCl salt diet for 2 weeks. Results: A high-salt diet for 2 weeks generated higher blood pressure in WT mice but not in Fabry disease mice. Free water clearance (FWC) and electrolyte free water clearance (EFWC) in groups fed with a high salt diet were lower than those in groups fed with a normal salt diet. Both high salt-fed WT and Fabry disease mice had decreased gamma-subunit of epithelial Na channel (ENaC) compared with normal salt-fed groups. In Fabry disease mice fed a high-salt diet, there was an decrease in renal expressions of Na+/H+ exchanger isoform 3 (NHE3) and Na+-Cl- cotransporter (NCC) compared with WT mice fed a high-salt diet. Although renal expression of vasopressin V2 receptor (V2R) was upregulated in Fabry mice fed with a high salt diet, renal aquaporin 2 (AQP2) level failed to increase up to the level in WT mice fed with a high salt diet. Conclusion: These findings suggest that the decreased expression of NHE3 and NCC and impaired response of AQP2 in kidneys of Fabry disease to a salt load could be one of mechanisms by which Fabry disease could have resistance to the development of hypertension.

In Vivo Delivery of Therapeutic Molecules by Transplantation of Genome-Edited Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (iPSCs) have already been used in transplantation therapies. Currently, cells from healthy people are transplanted into patients with diseases. With the rapid evolution of genome editing technology, genetic modification could be applied to enhance the therapeutic effects of iPSCs, such as the introduction of secreted molecules to make the cells a drug delivery system. Here, we addressed this possibility by utilizing a Fabry disease mouse model, as a proof of concept. Fabry disease is caused by the lack of α-galactosidase A (GLA). We previously developed an immunotolerant therapeutic molecule, modified α-N-acetylgalactosaminidase (mNAGA). We confirmed that secreted mNAGA from genome-edited iPSCs compensated for the GLA activity in GLA-deficient cells using an in vitro co-culture system. Moreover, iPSCs transplanted into Fabry model mice secreted mNAGA and supplied GLA activity to the liver. This study demonstrates the great potential of genome-edited iPSCs secreting therapeutic molecules.

Fabry Disease Mouse Is Resistant to High-Salt Diet-Induced Hypertension Probably via Dysfunctional Sodium Transporters and Aquaporin 2

Fabry Disease Mouse Is Resistant to High-Salt Diet-Induced Hypertension Probably via Dysfunctional Sodium Transporters and Aquaporin 2

CARS Imaging Advances Early Diagnosis of Cardiac Manifestation of Fabry Disease.

Vibrational spectroscopy can detect characteristic biomolecular signatures and thus has the potential to support diagnostics. Fabry disease (FD) is a lipid disorder disease that leads to accumulations of globotriaosylceramide in different organs, including the heart, which is particularly critical for the patient’s prognosis. Effective treatment options are available if initiated at early disease stages, but many patients are late- or under-diagnosed. Since Coherent anti-Stokes Raman (CARS) imaging has a high sensitivity for lipid/protein shifts, we applied CARS as a diagnostic tool to assess cardiac FD manifestation in an FD mouse model. CARS measurements combined with multivariate data analysis, including image preprocessing followed by image clustering and data-driven modeling, allowed for differentiation between FD and control groups. Indeed, CARS identified shifts of lipid/protein content between the two groups in cardiac tissue visually and by subsequent automated bioinformatic discrimination with a mean sensitivity of 90–96%. Of note, this genotype differentiation was successful at a very early time point during disease development when only kidneys are visibly affected by globotriaosylceramide depositions. Altogether, the sensitivity of CARS combined with multivariate analysis allows reliable diagnostic support of early FD organ manifestation and may thus improve diagnosis, prognosis, and possibly therapeutic monitoring of FD.

α-Galactosidase A Augmentation by Non-Viral Gene Therapy: Evaluation in Fabry Disease Mice.

Fabry disease (FD) is a monogenic X-linked lysosomal storage disorder caused by a deficiency in the lysosomal enzyme α-Galactosidase A (α-Gal A). It is a good candidate to be treated with gene therapy, in which moderately low levels of enzyme activity should be sufficient for clinical efficacy. In the present work we have evaluated the efficacy of a non-viral vector based on solid lipid nanoparticles (SLN) to increase α-Gal A activity in an FD mouse model after intravenous administration. The SLN-based vector incremented α-Gal A activity to about 10%, 15%, 20% and 14% of the levels of the wild-type in liver, spleen, heart and kidney, respectively. In addition, the SLN-based vector significantly increased α-Gal A activity with respect to the naked pDNA used as a control in plasma, heart and kidney. The administration of a dose per week for three weeks was more effective than a single-dose administration. Administration of the SLN-based vector did not increase liver transaminases, indicative of a lack of toxicity. Additional studies are necessary to optimize the efficacy of the system; however, these results reinforce the potential of lipid-based nanocarriers to treat FD by gene therapy.

Fabry disease exacerbates renal interstitial fibrosis after unilateral ureteral obstruction via impaired autophagy and enhanced apoptosis.

BackgroundFabry disease is a rare X-linked genetic lysosomal disorder caused by mutations in the GLA gene encoding alpha-galactosidase A. Despite some data showing that profibrotic and proinflammatory cytokines and oxidative stress could be involved in Fabry disease-related renal injury, the pathogenic link between metabolic derangement within cells and renal injury remains unclear.MethodsRenal fibrosis was triggered by unilateral ureteral obstruction (UUO) in mice with Fabry disease to investigate the pathogenic mechanism leading to fibrosis in diseased kidneys.ResultsCompared to kidneys of wild-type mice, lamellar inclusion bodies were recognized in proximal tubules of mice with Fabry disease. Sirius red and trichrome staining revealed significantly increased fibrosis in all UUO kidneys, though it was more prominent in obstructed Fabry kidneys. Renal messenger RNA levels of inflammatory cytokines and profibrotic factors were increased in all UUO kidneys compared to sham-operated kidneys but were not significantly different between UUO control and UUO Fabry mice. Protein levels of Nox2, Nox4, NQO1, catalase, SOD1, SOD2, and Nrf2 were not significantly different between UUO control and UUO Fabry kidneys, while the protein contents of LC3-II and LC3-I and expression of Beclin1 were significantly decreased in UUO kidneys of Fabry disease mouse models compared with wild-type mice. Notably, TUNEL-positive cells were elevated in obstructed kidneys of Fabry disease mice compared to wild-type control and UUO mice.ConclusionThese findings suggest that impaired autophagy and enhanced apoptosis are probable mechanisms involved in enhanced renal fibrosis under the stimulus of UUO in Fabry disease.

Assessing the role of glycosphingolipids in the phenotype severity of Fabry disease mouse model

Fabry disease is caused by deficient activity of α-galactosidase A, an enzyme that hydrolyzes the terminal α-galactosyl moieties from glycolipids and glycoproteins, and subsequent accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3), globotriaosylsphingosine (lyso-Gb3), and galabiosylceramide. However, there is no known link between these compounds and disease severity. In this study, we compared Gb3 isoforms (various fatty acids) and lyso-Gb3 analogs (various sphingosine modifications) in two strains of Fabry disease mouse models: a pure C57BL/6 (B6) background or a B6/129 mixed background, with the latter exhibiting more prominent cardiac and renal hypertrophy and thermosensation deficits. Total Gb3 and lyso-Gb3 levels in the heart, kidney, and dorsal root ganglion (DRG) were similar in the two strains. However, levels of the C20-fatty acid isoform of Gb3 and particular lyso-Gb3 analogs (+18, +34) were significantly higher in Fabry-B6/129 heart tissue when compared with Fabry-B6. By contrast, there was no difference in Gb3 and lyso-Gb3 isoforms/analogs in the kidneys and DRG between the two strains. Furthermore, using immunohistochemistry, we found that Gb3 massively accumulated in DRG mechanoreceptors, a sensory neuron subpopulation with preserved function in Fabry disease. However, Gb3 accumulation was not observed in nonpeptidergic nociceptors, the disease-relevant subpopulation that has remarkably increased isolectin-B4 (the marker of nonpeptidergic nociceptors) binding and enlarged cell size. These findings suggest that specific species of Gb3 or lyso-Gb3 may play major roles in the pathogenesis of Fabry disease, and that Gb3 and lyso-Gb3 are not responsible for the pathology in all tissues or cell types.

Systemic Treatment of Fabry Disease Using a Novel AAV9 Vector Expressing α-Galactosidase A

Fabry disease is a rare X-linked disorder affecting α-galactosidase A, a rate-limiting enzyme in lysosomal catabolism of glycosphingolipids. Current treatments present important limitations, such as low half-life and limited distribution, which gene therapy can overcome. The aim of this work was to test a novel adeno-associated viral vector, serotype 9 (AAV9), ubiquitously expressing human α-galactosidase A to treat Fabry disease (scAAV9-PGK-GLA). The vector was preliminary tested in newborns of a Fabry disease mouse model. 5 months after treatment, α-galactosidase A activity was detectable in the analyzed tissues, including the central nervous system. Moreover, we tested the vector in adult animals of both sexes at two doses and disease stages (presymptomatic and symptomatic) by single intravenous injection. We found that the exogenous α-galactosidase A was active in peripheral tissues as well as the central nervous system and prevented glycosphingolipid accumulation in treated animals up to 5 months following injection. Antibodies against α-galactosidase A were produced in 9 out of 32 treated animals, although enzyme activity in tissues was not significantly affected. These results demonstrate that scAAV9-PGK-GLA can drive widespread and sustained expression of α-galactosidase A, cross the blood brain barrier after systemic delivery, and reduce pathological signs of the Fabry disease mouse model.

Characterization of the microglia in three non-overlapping lysosomal storage diseases

Abstract Lysosomal storage diseases (LSDs) are a large family of inherited disorders characterized by abnormal endolysosomal accumulation of cellular material due to catabolic enzyme or transporter deficiencies. Depending on the affected metabolic pathway, these diseases manifest with somatic or central nervous system signs and symptoms. Neuroinflammation is a hallmark feature of LSDs with neurologic involvement such as mucolipidosis type IV (Mcoln1) or Niemann-Pick disease, type C1 (Npc1), but not Fabry disease (Gla). We investigated the properties of microglia from these three disorders using mouse models and a combination of flow cytometric, RNA sequencing, biochemical and immunofluorescence analyses. Stored material is present in the microglia of the different mice however only in Niemann-Pick disease, type C1 did we observe that neuroinflammation increases with disease progression. Fabry disease mice do not have neurological manifestation, and this is reflected by the minor changes in microglial properties. Lastly, mucolipidosis type IV mice have an early gliosis which decreases with disease progression. Comparison among microglia transcriptome profiles form these 3 diseases, and other disorders where transcriptome data is available, showed similarities with other neurodegenerative diseases. The phenotypic similarities between microglia from rare monogenic disorders, where the primary metabolic disturbance is known, and common neuroinflammatory diseases may provide novel insights for therapeutic intervention.

Determination of globotriaosylceramide analogs in the organs of a mouse model of Fabry disease

Fabry disease is a heritable lipid disorder caused by the low activity of α-galactosidase A and characterized by the systemic accumulation of globotriaosylceramide (Gb3). Recent studies have reported a structural heterogeneity of Gb3 in Fabry disease, including Gb3 isoforms with different fatty acids and Gb3 analogs with modifications on the sphingosine moiety. However, Gb3 assays are often performed only on the selected Gb3 isoforms. To precisely determine the total Gb3 concentration, here we established two methods for determining both Gb3 isoforms and analogs. One was the deacylation method, involving Gb3 treatment with sphingolipid ceramide N-deacylase, followed by an assay of the deacylated products, globotriaosylsphingosine (lyso-Gb3) and its analogs, by ultra-performance LC coupled to tandem MS (UPLC-MS/MS). The other method was a direct assay established in the present study for 37 Gb3 isoforms and analogs/isoforms by UPLC-MS/MS. Gb3s from the organs of symptomatic animals of a Fabry disease mouse model were mainly Gb3 isoforms and two Gb3 analogs, such as Gb3(+18) containing the lyso-Gb3(+18) moiety and Gb3(−2) containing the lyso-Gb3(−2) moiety. The total concentrations and Gb3 analog distributions determined by the two methods were comparable. Gb3(+18) levels were high in the kidneys (24% of total Gb3) and the liver (13%), and we observed Gb3(−2) in the heart (10%) and the kidneys (5%). These results indicate organ-specific expression of Gb3 analogs, insights that may lead to a deeper understanding of the pathophysiology of Fabry disease.

Unique molecular signature in mucolipidosis type IV microglia.

BackgroundLysosomal storage diseases (LSD) are a large family of inherited disorders characterized by abnormal endolysosomal accumulation of cellular material due to catabolic enzyme and transporter deficiencies. Depending on the affected metabolic pathway, LSD manifest with somatic or central nervous system (CNS) signs and symptoms. Neuroinflammation is a hallmark feature of LSD with CNS involvement such as mucolipidosis type IV, but not of others like Fabry disease.MethodsWe investigated the properties of microglia from LSD with and without major CNS involvement in 2-month-old mucolipidosis type IV (Mcoln1−/−) and Fabry disease (Glay/−) mice, respectively, by using a combination of flow cytometric, RNA sequencing, biochemical, in vitro and immunofluorescence analyses.ResultsWe characterized microglia activation and transcriptome from mucolipidosis type IV and Fabry disease mice to determine if impaired lysosomal function is sufficient to prime these brain-resident immune cells. Consistent with the neurological pathology observed in mucolipidosis type IV, Mcoln1−/− microglia demonstrated an activation profile with a mixed neuroprotective/neurotoxic expression pattern similar to the one we previously observed in Niemann-Pick disease, type C1, another LSD with significant CNS involvement. In contrast, the Fabry disease microglia transcriptome revealed minimal alterations, consistent with the relative lack of CNS symptoms in this disease. The changes observed in Mcoln1−/− microglia showed significant overlap with alterations previously reported for other common neuroinflammatory disorders including Alzheimer’s, Parkinson’s, and Huntington’s diseases. Indeed, our comparison of microglia transcriptomes from Alzheimer’s disease, amyotrophic lateral sclerosis, Niemann-Pick disease, type C1 and mucolipidosis type IV mouse models showed an enrichment in “disease-associated microglia” pattern among these diseases.ConclusionsThe similarities in microglial transcriptomes and features of neuroinflammation and microglial activation in rare monogenic disorders where the primary metabolic disturbance is known may provide novel insights into the immunopathogenesis of other more common neuroinflammatory disorders.Trial registrationClinicalTrials.gov, NCT01067742, registered on February 12, 2010

The glycosylation design space for recombinant lysosomal replacement enzymes produced in CHO cells

Lysosomal replacement enzymes are essential therapeutic options for rare congenital lysosomal enzyme deficiencies, but enzymes in clinical use are only partially effective due to short circulatory half-life and inefficient biodistribution. Replacement enzymes are primarily taken up by cell surface glycan receptors, and glycan structures influence uptake, biodistribution, and circulation time. It has not been possible to design and systematically study effects of different glycan features. Here we present a comprehensive gene engineering screen in Chinese hamster ovary cells that enables production of lysosomal enzymes with N-glycans custom designed to affect key glycan features guiding cellular uptake and circulation. We demonstrate distinct circulation time and organ distribution of selected glycoforms of α-galactosidase A in a Fabry disease mouse model, and find that an α2-3 sialylated glycoform designed to eliminate uptake by the mannose 6-phosphate and mannose receptors exhibits improved circulation time and targeting to hard-to-reach organs such as heart. The developed design matrix and engineered CHO cell lines enables systematic studies towards improving enzyme replacement therapeutics.