Safety and Efficacy of Intravenous and Intrathecal Delivery of AAV9-Mediated ARSA in Minipigs.

Metachromatic leukodystrophy (MLD) is a hereditary neurodegenerative disease characterized by demyelination and motor and cognitive impairments due to deficiencies of the lysosomal enzyme arylsulfatase A (ARSA) or the saposin B activator protein (SapB). Current treatments are limited; however, gene therapy using adeno-associated virus (AAV) vectors for ARSA delivery has shown promising results. The main challenges for MLD gene therapy include optimizing the AAV dosage, selecting the most effective serotype, and determining the best route of administration for ARSA delivery into the central nervous system. This study aims to evaluate the safety and efficacy of AAV serotype 9 encoding ARSA (AAV9-ARSA) gene therapy when administered intravenously or intrathecally in minipigs, a large animal model with anatomical and physiological similarities to humans. By comparing these two administration methods, this study contributes to the understanding of how to improve the effectiveness of MLD gene therapy and offers valuable insights for future clinical applications.

Atidarsagene autotemcel for metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is a rare autosomal recessive disorder of sphingolipid metabolism, due to a deficiency of the enzyme arylsulfatase A (ARSA). The main clinical signs of the disease are secondary to central and peripheral nervous system demyelination. MLD is subdivided into early- and late-onset subtypes based upon the onset of neurological disease. The early-onset subtype is associated with a more rapid progression of the disease that leads to death within the first decade of life. Until recently, no effective treatment was available for MLD. The blood-brain barrier (BBB) prevents systemically administered enzyme replacement therapy from reaching target cells in MLD. The evidence for the efficacy of hematopoietic stem cell transplantation is limited to the late-onset MLD subtype. Here, we review the preclinical and clinical studies that facilitated the approval of the ex vivo gene therapy atidarsagene autotemcel for early-onset MLD by the European Medicines Agency (EMA) in December 2020. This approach was studied in an animal model first and then in a clinical trial, eventually proving its efficacy in preventing disease manifestations in presymptomatic patients and stabilizing its progression in paucisymptomatic subjects. This new therapeutic consists of patients' CD34+ hematopoietic stem/progenitor cells (HSPCs) transduced with a lentiviral vector encoding functional ARSA cDNA. The gene-corrected cells get reinfused into the patients after a cycle of chemotherapy conditioning.

Treatment of adult metachromatic leukodystrophy model mice using intrathecal administration of type 9 AAV vector encoding arylsulfatase A.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by an arylsulfatase A (ARSA) deficiency and characterized by severe neurological symptoms resulting from demyelination within the central and peripheral nervous systems. We investigated the feasibility and efficacy of intrathecal administration of a type 9 adeno-associated viral vector encoding ARSA (AAV9/ARSA) for the treatment of 6-week-old MLD model mice, which are presymptomatic, and 1-year-old mice, which exhibit neurological abnormalities. Immunohistochemical analysis following AAV9/ARSA administration showed ARSA expression within the brain, with highest activities in the cerebellum and olfactory bulbs. In mice treated at 1 year, alcian blue staining and quantitative analysis revealed significant decreases in stored sulfatide. Behaviorally, mice treated at 1 year showed no improvement in their ability to traverse narrow balance beams as compared to untreated mice. By contrast, MLD mice treated at 6 weeks showed significant decreases in stored sulfatide throughout the entire brain and improved ability to traverse narrow balance beams. These findings suggest intrathecal administration of an AAV9/ARSA vector is a promising approach to treating genetic diseases of the central nervous system, including MLD, though it may be essential to begin therapy before the onset of neurological symptoms.

A dual-enzyme cleavable linker for antibody-drug conjugates.

A novel enzyme cleavable linker for antibody-drug conjugates is reported. The 3-O-sulfo-ß-galactose linker is cleaved sequentially by two lysosomal enzymes - arylsulfatase A and ß-galactosidase - to release the payload in targeted cells. An α-HER2 antibody-drug conjugate synthesised using this highly hydrophilic dual-cleavable linker exhibited excellent cytotoxicity and selectivity.

Brain cell type-specific endocytosis of arylsulfatase A identifies limitations of enzyme-based therapies for metachromatic leukodystrophy.

Enzyme replacement therapies, allogeneic bone marrow transplantation and gene therapies are treatment options for lysosomal storage diseases caused by inherited deficiencies of soluble lysosomal enzymes. Independent from the approach, the enzyme must be delivered to lysosomes of deficient patient cells. Little is known about the dissemination of enzyme within a tissue where cells compete for uptake via different receptor systems, binding affinities and endocytic rates. To evaluate dissemination and lysosomal targeting of a lysosomal enzyme in the CNS, we analysed receptor-mediated endocytosis of arylsulfatase A (ASA) by different types of brain-derived cell lines and primary murine brain cells. For ASA expressed by chinese hamster ovary cells for enzyme replacement therapy of metachromatic leukodystrophy, endocytic rates decline from microglia to neurons and astrocytes and to oligodendrocytes. Only immature oligodendrocytes endocytose significant amounts of enzyme. Uptake by non-microglial cells is due to mannose 6-phosphate receptors, whereas several receptor systems participate in endocytosis by microglial cells. Interestingly, ASA expressed by microglial cells cannot be taken up in a mannose 6-phosphate dependent manner. The resulting failure to correct non-microglial cells corroborates in vivo data and indicates that therapeutic effects of allogeneic bone marrow transplantation and hematopoietic stem cell gene therapy on metachromatic leukodystrophy are independent of metabolic cross-correction of neurons, astrocytes and oligodendrocytes by receptor-mediated endocytosis.

Arylsulfatases A and B: From normal tissues to malignant tumors.

Arylsulfatases are lysosomal enzymes with important roles in the cell metabolism. Several subtypes of arylsulfatase are known, from A to K. Congenital deficiencies of arylsulfatases, especially A (ARSA) and B (ARSB), can induce metabolic disorders such as metachromatic leucodystrophy (ARSA deficiency) and Maroteaux-Lamy syndrome (ARSB deficiency). ARSA and ARSB pseudodeficiencies were recently described but their exact roles are far to be known. The aim of this review was to synthesize the literature data, combined with personal results, regarding the roles of ARSA and ARSB in non-tumor disorders but also carcinogenesis. Few than 50 published papers regard ARSA and ARSB expression in cancer. They suggest decreased activity of these arylsulfatases in most of carcinomas, compared with normal tissues. However, the clinical impact is still unknown. Further complex studies are necessary to be done, to understand the role of ARSA and ARSB expression in cancer.

Measurement of recombinant human arylsulfatase A and leukocyte sulfatase activities by analytical isotachophoresis.

Metachromatic Leukodystrophy (MLD) and Multiple Sulfatase Deficiency (MSD) are rare and ultra-rare lysosomal storage diseases. Due to enzyme defects, patients are unable to split the sulfategroup from the respective substrates. In MSD all sulfatases are affected due to a defect of the Sulfatase Modifying Factor 1 (SUMF1) gene coding for the formylglycine generating enzyme (FGE) necessary for the modification of the active site of sulfatases. In MLD mutations in the arylsulfatase A (ARSA) gene cause ARSA deficiency with subsequent accumulation of 3-sulfogalactocerebroside especially in oligodendrocytes. The clinical consequence is demyelination and a devastating neurological disease. Enzyme replacement therapy (ERT) with recombinant human arylsulfatase A (rhARSA), gene therapy, and stem cell transplantation are suggested as new therapeutic options. The aim of our study was to characterize rhARSA concerning its substrate specificity using analytical isotachophoresis (ITP). Substrate specificity could be demonstrated by sulfate splitting from the natural substrates 3-sulfogalactocerebroside and ascorbyl-2-sulfate and the artificial substrate p-nitrocatecholsulfate, whereas galactose-6-sulfate, a substrate of galactose-6­sulfurylase, was totally resistant. In contrast, leukocyte extracts of healthy donors were able to split sulfate also from galactose-6-sulfate. The ITP method allows therefore a rapid and simple differentiation between samples of MLD and MSD patients and healthy donors. Therefore, the isotachophoretic diagnostic assay from leukocyte extracts described here provides a fast and efficient way for the diagnosis of MLD and MSD patients and an elegant system to differentiate between these diseases in one assay.

Enzymatic characterization of novel arylsulfatase A variants using human arylsulfatase A-deficient immortalized mesenchymal stromal cells.

Metachromatic leukodystrophy (MLD) is an autosomal-recessive lysosomal storage disease caused by mutations in the ARSA gene leading to arylsulfatase A (ARSA) deficiency and causing sulfatide accumulation. Main symptoms of the disease are progressive demyelination, neurological dysfunction, and reduced life expectancy. To date, more than 200 different ARSA variants have been reported in MLD patients. Here, we report the biochemical characterization of seven novel pathogenic variants (c.98T > C, c.195delC, c.229G > C, c.545C > G, c.674A > G, c.852T > A, and c.1274A > G), which were found when sequencing a cohort of 31 German MLD families. For that purpose, the ARSA cDNAs carrying the respective mutations inserted by site-directed mutagenesis were cloned into a MigR1 (MSCV, IRES, GFP, retrovirus-1) vector. The constructs were overexpressed using retroviral gene transfer in immortalized, human multipotent mesenchymal stromal cells prepared from a patient deficient in ARSA activity (late infantile MLD). In this novel ARSA-/- cell system, the seven ARSA mutants showed ARSA activity of less than 10% when compared with wild type, which is evidence for the pathogenicity of all seven variants. In conclusion, the system of ARSA-/- -immortalized MSC turned out to be a helpful novel tool for the biochemical characterization of ARSA variants.

Metachromatic Leukodystrophy (MLD): a Pakistani Family with Novel ARSA Gene Mutation.

A deficiency of the enzyme arylsulfatase A (ARSA) causes a progressive neurodegenerative lysosomal storage disease known as metachromatic leukodystrophy (MLD). Diagnosis is based on the onset of neurological symptoms, presence of gait abnormalities, spasticity, decreased muscle stretch reflexes and neuro-radiological evidence of demyelination. The purpose of the present study was to identify any mutation in the candidate ARSA gene in a family of late infantile MLD patients. The diagnosis of suspected MLD patients was confirmed by a MRI report and low ARSA enzymatic activity in leukocytes. Sanger sequencing of full-length coding regions of ARSA gene was performed. Changes in the nucleotide sequence were determined by comparing the obtained data with the wild-type sequence. mRNA expression was analysed using real-time PCR. A novel base pair substitution at position c.338T>C (p.L113P) of ARSA gene was observed in the family and was confirmed in a normal population via ARMS-PCR and Sanger sequencing. The mRNA expression of ARSA gene showed a significant difference between normal and carrier individuals (p = 0.0008). In silico analysis by POLYPHEN, a pathogenicity prediction tool, predicted the possible damaging nature of this mutation. I-TASSER, a protein-modelling server, demonstrated the effects of this mutation on different domains of the ARSA protein, which plays a crucial role in the structural and functional integrity of enzyme. The novel p.L113P mutation in a Pakistani family with late infantile MLD has a pathogenic and destructive effect on the protein structure and function of ARSA. It is the first case reported in a Pakistani population using genetic analysis.

Arylsulfatase A Overexpressing Human iPSC-derived Neural Cells Reduce CNS Sulfatide Storage in a Mouse Model of Metachromatic Leukodystrophy.

Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disorder resulting from a functional deficiency of arylsulfatase A (ARSA), an enzyme that catalyzes desulfation of 3-O-sulfogalactosylceramide (sulfatide). Lack of active ARSA leads to the accumulation of sulfatide in oligodendrocytes, Schwann cells and some neurons and triggers progressive demyelination, the neuropathological hallmark of MLD. Several therapeutic approaches have been explored, including enzyme replacement, autologous hematopoietic stem cell-based gene therapy, intracerebral gene therapy or cell-based gene delivery into the central nervous system (CNS). However, long-term treatment of the blood-brain-barrier protected CNS remains challenging. Here we used MLD patient-derived induced pluripotent stem cells (iPSCs) to generate long-term self-renewing neuroepithelial stem cells and astroglial progenitors for cell-based ARSA replacement. Following transplantation of ARSA-overexpressing precursors into ARSA-deficient mice we observed a significant reduction of sulfatide storage up to a distance of 300 µm from grafted cells. Our data indicate that neural precursors generated via reprogramming from MLD patients can be engineered to ameliorate sulfatide accumulation and may thus serve as autologous cell-based vehicle for continuous ARSA supply in MLD-affected brain tissue.

Intracerebral Gene Therapy Using AAVrh.10-hARSA Recombinant Vector to Treat Patients with Early-Onset Forms of Metachromatic Leukodystrophy: Preclinical Feasibility and Safety Assessments in Nonhuman Primates.

No treatment is available for early-onset forms of metachromatic leukodystrophy (MLD), a lysosomal storage disease caused by autosomal recessive defect in arylsulfatase A (ARSA) gene causing severe demyelination in central and peripheral nervous systems. We have developed a gene therapy approach, based on intracerebral administration of AAVrh.10-hARSA vector, coding for human ARSA enzyme. We have previously demonstrated potency of this approach in MLD mice lacking ARSA expression. We describe herein the preclinical efficacy, safety, and biodistribution profile of intracerebral administration of AAVrh.10-hARSA to nonhuman primates (NHPs). NHPs received either the dose planned for patients adjusted to the brain volume ratio between child and NHP (1×dose, 1.1×10(11) vg/hemisphere, unilateral or bilateral injection) or 5-fold this dose (5×dose, 5.5×10(11) vg/hemisphere, bilateral injection). NHPs were subjected to clinical, biological, and brain imaging observations and were euthanized 7 or 90 days after injection. There was no toxicity based on clinical and biological parameters, nor treatment-related histological findings in peripheral organs. A neuroinflammatory process correlating with brain MRI T2 hypersignals was observed in the brain 90 days after administration of the 5×dose, but was absent or minimal after administration of the 1×dose. Antibody response to AAVrh.10 and hARSA was detected, without correlation with brain lesions. After injection of the 1×dose, AAVrh.10-hARSA vector was detected in a large part of the injected hemisphere, while ARSA activity exceeded the normal endogenous activity level by 14-31%. Consistently with other reports, vector genome was detected in off-target organs such as liver, spleen, lymph nodes, or blood, but not in gonads. Importantly, AAVrh.10-hARSA vector was no longer detectable in urine at day 7. Our data demonstrate requisite safe and effective profile for intracerebral AAVrh.10-hARSA delivery in NHPs, supporting its clinical use in children affected with MLD.

Comparative efficacy and safety of multiple routes of direct CNS administration of adeno-associated virus gene transfer vector serotype rh.10 expressing the human arylsulfatase A cDNA to nonhuman primates.

Metachromatic leukodystrophy (MLD), a fatal disorder caused by deficiency of the lysosomal enzyme arylsulfatase A (ARSA), is associated with an accumulation of sulfatides, causing widespread demyelination in both central and peripheral nervous systems. On the basis of prior studies demonstrating that adeno-associated virus AAVrh.10 can mediate widespread distribution in the CNS of a secreted lysosomal transgene, and as a prelude to human trials, we comparatively assessed the optimal CNS delivery route of an AAVrh.10 vector encoding human ARSA in a large animal model for broadest distribution of ARSA enzyme. Five routes were tested (each total dose, 1.5 × 10(12) genome copies of AAVrh.10hARSA-FLAG): (1) delivery to white matter centrum ovale; (2) deep gray matter delivery (putamen, thalamus, and caudate) plus overlying white matter; (3) convection-enhanced delivery to same deep gray matter locations; (4) lateral cerebral ventricle; and (5) intraarterial delivery with hyperosmotic mannitol to the middle cerebral artery. After 13 weeks, the distribution of ARSA activity subsequent to each of the three direct intraparenchymal administration routes was significantly higher than in phosphate-buffered saline-administered controls, but administration by the intraventricular and intraarterial routes failed to demonstrate measurable levels above controls. Immunohistochemical staining in the cortex, white matter, deep gray matter of the striatum, thalamus, choroid plexus, and spinal cord dorsal root ganglions confirmed these results. Of the five routes studied, administration to the white matter generated the broadest distribution of ARSA, with 80% of the brain displaying more than a therapeutic (10%) increase in ARSA activity above PBS controls. No significant toxicity was observed with any delivery route as measured by safety parameters, although some inflammatory changes were seen by histopathology. We conclude that AAVrh.10-mediated delivery of ARSA via CNS administration into the white matter is likely to be safe and yields the widest distribution of ARSA, making it the most suitable route of vector delivery.

[Effects of rutin on the activity of antioxidant enzymes and xenobiotic-metabolizing enzymes in liver of rats fed diets with different level of fat].

The study has been carried out on 6 groups of male Wistar rats, which received semi-synthetic diets within 28 days. Rats of 1st and 4th group received fat-free diet, 2nid.and 5th - diet containing standard amount of fat (10% by weight, 26% by caloric content; lard/sunflower oil - 1/1); 3rd and 6th group - a high-fat diet (30% by weight, 56% by caloric content). During the last 14 days of the experiment rats received rutin in the dose of 40 mg/kg b.w. AOA, MDA level and the activity of paraoxonase I have been evaluated in blood serum. In rat liver along with the parameters of the antioxidant status (MDA level, activity of paraoxonase 1, quinone reductase, heme oxygenase-1) the activity of xenobiotic-metabolizing enzymes (XME) (CYP1A1, CYP1A2, CYP3A1, CYP2B1, UDP-glucuronosyl transferase and glutathione transferase) and the activity of lysosomal enzymes (arylsulfatase A and B, ß-galactosidase and ß-glucuronidase) have been investigated. Elevation of the activity of antioxidant enzymes and XME in liver with the increase of diet fat content has been-noted. Rutin admihistration had no effect onparamete6rs of antioxidant status and decreased unsedimentable activity of lysosomal enzymes that did not depend on fat content in the diet. Rutin receiving increased the activity of all studied XME in rats fed standard diet, but practically did not effect on their activity in rats fed by fat-free and high-fat diets. Thus, rutin in pharmacological dose has no effect on the activity of antioxidant enzymes that doesn't depend on the level of fat in the diet, while the decrease or increase of diet fat content modulates (weakens) the influence of rutin on the XME activity.

Promotion of autophagy at the maturation step by IL-6 is associated with the sustained mitogen-activated protein kinase/extracellular signal-regulated kinase activity.

Increased autophagic vacuoles (AVs) occur in injured or degenerating neurons, under both developmental and pathological situations. Although an induced autophagy has been shown in inflammation response to cell factors, the underlying mechanism(s) remain(s) unknown. Here, we show that both cell factor IL-6 and environmental toxin MPP(+) promote the formation of vacuolation in SHSY5Y cells. By electron and immunofluorescent microscopy analyses, we showed that these structures are acid autolysosomes, containing cellular debris, and labeled by LC3 or LAMP1, markers of autophagosomes or lysosomes, respectively. Combining MPP(+) and IL-6 do not further increase vacuolation of SHSY5Y cells, and the vacuolation is less than that in the MPP(+)-treated group. MPP(+)-induced vacuolation results from significant increase in autophagy formation and delay in autophagy degradation, in relation to a decline of the lysosomal activity of arylsulfatase A. At molecular level, we show that this defect in autolysosomal maturation is independent of mammalian target of rapamycin and p38 inhibitions. Most importantly, we provide the first evidence that activation of ERK pathway is sufficient to commit cell to autophagic vacuolation. The sustained activation is required for MPP(+) to disrupt the autophagic pathway. IL-6 also induces a temporary and significant activation of ERK, but not sustained activation, and change sustained activation in MPP(+)-treated group into temporary activation. Taken together, these findings strongly support that IL-6 promotes the maturation of autophagosomes into functional autolysosomes by regulating ERK.

Can endometrial arylsulfatase A activity predict the onset of endometrial polyps over the years?

PURPOSE: To assess if arylsulfatase A activity (ASA) and sulfatide (SL) concentration in the human endometrium can be predictive of the development of endometrial polyps over the years, since ASA activity reflects the endometrial sensitivity to hormones. METHODS: ASA activity and SL concentration were determined by biochemical procedures on endometrial samples collected between 1990 and 1994 in non-menopausal women. These women underwent a new endometrial sampling following the clinical indication some years after the first endometrial sampling. The histological assessment of the second endometrial specimens found four patients with normal endometrial pattern and 10 patients with one or more endometrial polyps. ASA activity/years elapsed and SL concentration/years elapsed were compared using two tailed Mann-Whitney test for unpaired data between patients with normal pattern and patients with endometrial polyps. RESULTS: Median ASA activities were 2.62 (normal pattern) versus 1.85 (endometrial polyps) nmol hydrolized substrate/min. Median activity/years elapsed is higher in patients with second endometrial sample presenting normal pattern (p=0.006) and median SL concentration/years elapsed does not differ significantly among groups, even if median SL concentration seems to be higher in patients who subsequently developed polyps (1031 µg/g of fresh tissue versus 341,5 µg/g of fresh tissue). CONCLUSIONS: ASA activity can predict the onset of endometrial polyps over the years.

Can endometrial arylsulfatase A activity predict the onset of endometrial polyps over the years? / A atividade da arilsulfatase endometrial A pode prever a aparição de pólipos endometriais ao longo dos anos?

PURPOSE: To assess if arylsulfatase A activity (ASA) and sulfatide (SL) concentration in the human endometrium can be predictive of the development of endometrial polyps over the years, since ASA activity reflects the endometrial sensitivity to hormones. METHODS: ASA activity and SL concentration were determined by biochemical procedures on endometrial samples collected between 1990 and 1994 in non-menopausal women. These women underwent a new endometrial sampling following the clinical indication some years after the first endometrial sampling. The histological assessment of the second endometrial specimens found four patients with normal endometrial pattern and 10 patients with one or more endometrial polyps. ASA activity/years elapsed and SL concentration/years elapsed were compared using two tailed Mann-Whitney test for unpaired data between patients with normal pattern and patients with endometrial polyps. RESULTS: Median ASA activities were 2.62 (normal pattern) versus 1.85 (endometrial polyps) nmol hydrolized substrate/min. Median activity/years elapsed is higher in patients with second endometrial sample presenting normal pattern (p=0.006) and median SL concentration/years elapsed does not differ significantly among groups, even if median SL concentration seems to be higher in patients who subsequently developed polyps (1031 µg/g of fresh tissue versus 341,5 µg/g of fresh tissue). CONCLUSIONS: ASA activity can predict the onset of endometrial polyps over the years.

OBJETIVO: Avaliar se a atividade da arilsulfatase A (ASA) e a concentração de sulfatida (SL) no endométrio humano pode ser preditivo em relação ao desenvolvimento de pólipos endometriais ao longo dos anos, posto que atividade da ASA reflete a sensibilidade do endométrio aos hormônios. MÉTODOS: A atividade da ASA, assim como a concentração de SL, foi determinada por meio de procedimentos bioquímicos em amostras de endométrio coletadas entre 1990 e 1994, em mulheres que não se encontravam na menopausa. Essas mulheres foram submetidas a uma nova amostragem endometrial após indicação clínica alguns anos depois da primeira amostragem endometrial. A avaliação histológica dos segundos espécimes endometriais permitiu identificar quatro pacientes com padrão endometrial normal e 10 com um ou mais pólipos endometriais. A atividade da ASA/anos depois e a concentração de SL/anos depois foram comparadas, utilizando o teste bilateral U de Mann-Whitney para dados não pareados entre as pacientes com padrão normal e as pacientes com pólipos endometriais. RESULTADOS: A ativitade da ASA foi 2,62 (padrão normal) em comparação com 1,85 (endometrial pólipos) de substrato hidrolisado/min. A atividade da ASA/anos depois é maior em pacientes com segunda amostra endometrial a apresentarem um padrão normal (p=0,006), e a concentração mediana de SL/anos depois não difere de forma significativa entre os grupos, apesar de a concentração mediana de SL parecer maior em pacientes que posteriormente desenvolveram pólipos (1031 µg/g de tecido fresco em comparação com 341,5 µg/g de tecido fresco). CONCLUSÕES: A atividade da ASA pode prever a aparição de pólipos endometriais ao longo dos anos.

Complete mapping of a cystine knot and nested disulfides of recombinant human arylsulfatase A by multi-enzyme digestion and LC-MS analysis using CID and ETD.

Cystine knots or nested disulfides are structurally difficult to characterize, despite current technological advances in peptide mapping with high-resolution liquid chromatography coupled with mass spectrometry (LC-MS). In the case of recombinant human arylsulfatase A (rhASA), there is one cystine knot at the C-terminal, a pair of nested disulfides at the middle, and two out of three unpaired cysteines in the N-terminal region. The statuses of these cysteines are critical structure attributes for rhASA function and stability that requires precise examination. We used a unique approach to determine the status and linkage of each cysteine in rhASA, which was comprised of multi-enzyme digestion strategies (from Lys-C, trypsin, Asp-N, pepsin, and PNGase F) and multi-fragmentation methods in mass spectrometry using electron transfer dissociation (ETD), collision induced dissociation (CID), and CID with MS(3) (after ETD). In addition to generating desired lengths of enzymatic peptides for effective fragmentation, the digestion pH was optimized to minimize the disulfide scrambling. The disulfide linkages, including the cystine knot and a pair of nested cysteines, unpaired cysteines, and the post-translational modification of a cysteine to formylglycine, were all determined. In the assignment, the disulfide linkages were Cys138-Cys154, Cys143-Cys150, Cys282-Cys396, Cys470-Cys482, Cys471-Cys484, and Cys475-Cys481. For the unpaired cysteines, Cys20 and Cys276 were free cysteines, and Cys51 was largely converted to formylglycine (>70%). A successful methodology has been developed, which can be routinely used to determine these difficult-to-resolve disulfide linkages, ensuring drug function and stability.

Efficacy of enzyme replacement therapy in an aggravated mouse model of metachromatic leukodystrophy declines with age.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by a functional deficiency of arylsulfatase A (ASA). Previous studies in ASA-knockout mice suggested enzyme replacement therapy (ERT) to be a promising treatment option. The mild phenotype of ASA-knockout mice did, however, not allow to examine therapeutic responses of the severe neurological symptoms that dominate MLD. We, therefore, generated an aggravated MLD mouse model displaying progressive demyelination and reduced nerve conduction velocity (NCV) and treated it by weekly intravenous injections of 20 mg/kg recombinant human ASA for 16 weeks. To analyze the stage-dependent therapeutic effects, ERT was initiated in a presymptomatic, early and progressed disease stage, at age 4, 8 and 12 months, respectively. Brain sulfatide storage, NCV and behavioral alterations were improved only in early, but not in late, treated mice showing a clear age-dependent efficacy of treatment. Hematopoietic stem cell transplantation (HSCT) for late-onset variants is the only therapeutic option for MLD to date. ERT resembles a part of the HSCT rationale, which is based on ASA supply by donor cells. Beyond ERT, our results, therefore, corroborate the clinical observation that HSCT is only effective when performed in early stages of disease.