Reduction Of Glycosaminoglycans Research Articles

Open-label, single-center, clinical study evaluating the safety, tolerability and clinical effects of pentosan polysulfate sodium in subjects with mucopolysaccharidosis I.

Lysosomal enzyme deficiency in mucopolysaccharidosis (MPS) I results in glycosaminoglycan (GAG) accumulation leading to pain and limited physical function. Disease-modifying treatments for MPS I, enzyme replacement, and hematopoietic stem cell therapy (HSCT), do not completely resolve MPS I symptoms, particularly skeletal manifestations. The GAG reduction, anti-inflammatory, analgesic, and tissue remodeling properties of pentosan polysulfate sodium (PPS) may provide disease-modifying treatment for musculoskeletal symptoms and joint inflammation in MPS I following ERT and/or HSCT. The safety and efficacy of PPS were evaluated in four subjects with MPS I aged 14-19 years, previously treated with ERT and/or HSCT. Subjects received doses of 0.75 mg/kg or 1.5 mg/kg PPS via subcutaneous injections weekly for 12 weeks, then every 2 weeks for up to 72 weeks. PPS was well tolerated at both doses with no serious adverse events. MPS I GAG fragment (UA-HNAc [1S]) levels decreased at 73 weeks. Cartilage degradation biomarkers serum C-telopeptide of crosslinked collagen (CTX) type I (CTX-I) and type II (CTX-II) and urine CTX-II decreased in all subjects through 73 weeks. PROMIS scores for pain interference, pain behavior, and fatigue decreased in all subjects through 73 weeks. Physical function, measured by walking distance and dominant hand function, improved at 49 and 73 weeks. Decreased GAG fragments and cartilage degradation biomarkers, and positive PROMIS outcomes support continued study of PPS as a potential disease-modifying treatment for MPS I with improved pain and function outcomes.

Hurler Syndrome Glycosaminoglycans Decrease in Cerebrospinal Fluid without Brain-Targeted Therapy.

Novel therapies for Hurler syndrome aim to cross the blood-brain barrier (BBB) to target neurodegeneration by degrading glycosaminoglycans (GAG). BBB penetration has been assumed with decreased cerebrospinal fluid (CSF) GAG, yet little is known about CSF GAG without brain-targeting therapies. We compared pre-transplant CSF GAG in patients who were treatment naïve (n = 19) versus receiving standard non-BBB penetrating enzyme replacement therapy (ERT, n = 12). In the ERT versus treatment naïve groups, CSF GAG was significantly lower across all content assayed, raising questions about using CSF GAG decrements to show BBB penetration. Future studies should compare GAG reduction in standard versus novel therapies. ANN NEUROL 2023;94:1182-1186.

CHARACTERIZATION OF NATIVE AND DECELLULARIZED PORCINE TENDON UNDER TENSION AND COMPRESSION: A CLOSER LOOK AT GLYCOSAMINOGLYCAN CONTRIBUTION TO TENDON MECHANICS

Decellularised porcine superflexor tendon (pSFT) has been demonstrated to be a suitable scaffold for anterior cruciate ligament reconstruction[1]. While the role of collagen in tendons is well known, the mechanical role of glycosaminoglycans (GAGs) is less clear and may be altered by the decellularisation process.To determine the effects of decellularisation on pSFT GAG content and mechanical function and to investigate the consequences of GAG loss in tensile and compressive loading.pSFTs were decellularised following previous techniques [2]. For GAG removal, native pSFTs were treated with chondroitinase ABC (ChABC; 0.1U/mL, 72h). Cell and GAG removal was validated using histology and quantitative assays. Native, decellularised and ChABC treated groups (n=6) were biomechanically characterised. In tension, specimens underwent stress relaxation and strength testing using previous protocols [1]. Stress relaxation data was fitted to a modified Maxwell-Weichert model to determine time-dependent (E1 & E2) and time-independent moduli (E0). The toe and linear region moduli (Etoe, Elinear), in addition to tensile strength (UTS) and failure strain were determined from strength testing. In compression, specimens underwent confined loading conditions (ramp at 10 s-1 to 10% strain and hold). The aggregate modulus (HA) and zero-strain permeability (k0) were determined using previous techniques [3]. Data was analysed by one-way ANOVA with Tukey post-hoc test to determine significant differences between test groups (p<0.05).Quantitative assays showed no GAG reduction post-decellularisation, but a significant reduction after ChABC treatment. HA was only significantly reduced in the ChABC group. k0 was significantly higher for the ChABC group compared to decellularised. E0 was significantly reduced in the decellularised group compared to native and ChABC groups, while E1 and E2 were not different between groups. Etoe, Elinear, UTS and failure strain were not different between groups.Decellularisation does not affect GAG content or impair mechanical function in pSFT. GAG loss adversely affects pSFT compressive properties, revealing major mechanical contribution under compression, but no significant role under tension.

Brain and visceral gene editing of mucopolysaccharidosis I mice by nasal delivery of the CRISPR/Cas9 system.

Mucopolysaccharidosis type I (MPS I) is an inherited disease caused by deficiency of the enzyme alpha-l-iduronidase (IDUA). MPS I affects several tissues, including the brain, leading to cognitive impairment in the severe form of the disease. Currently available treatments do not reach the brain. Therefore, in this study, we performed nasal administration (NA) of liposomal complexes carrying two plasmids encoding for the CRISPR/Cas9 system and for the IDUA gene targeting the ROSA26 locus, aiming at brain delivery in MPS I mice. Liposomes were prepared by microfluidization, and the plasmids were complexed to the formulations by adsorption. Physicochemical characterization of the formulations and complexes, in vitro permeation, and mucoadhesion in porcine nasal mucosa (PNM) were assessed. We performed NA repeatedly for 30 days in young MPS I mice, which were euthanized at 6months of age after performing behavioral tasks, and biochemical and molecular aspects were evaluated. Monodisperse mucoadhesive complexes around 110 nm, which are able to efficiently permeate the PNM. In animals, the treatment led to a modest increase in IDUA activity in the lung, heart, and brain areas, with reduction of glycosaminoglycan (GAG) levels in serum, urine, tissues, and brain cortex. Furthermore, treated mice showed improvement in behavioral tests, suggesting prevention of the cognitive damage. Nonviral gene editing performed through nasal route represents a potential therapeutic alternative for the somatic and neurologic symptoms of MPS I and possibly for other neurological disorders.

Chondroprotective effects of purple corn anthocyanins on advanced glycation end products induction through suppression of NF-\u03baB and MAPK signaling

Formation of advanced glycation end products (AGEs), which are associated with diabetes mellitus, contributes to prominent features of osteoarthritis, i.e., inflammation-mediated destruction of articular cartilage. Among the phytochemicals which play a role in anti-inflammatory effects, anthocyanins have also been demonstrated to have anti-diabetic properties. Purple corn is a source of three major anthocyanins: cyanidin-3-O-glucoside, pelargonidin-3-O-glucoside and peonidin-3-O-glucoside. Purple corn anthocyanins have been demonstrated to be involved in the reduction of diabetes-associated inflammation, suggesting that they may have a beneficial effect on diabetes-mediated inflammation of cartilage. This investigation of the chondroprotective effects of purple corn extract on cartilage degradation found a reduction in glycosaminoglycans released from AGEs induced cartilage explants, corresponding with diminishing of uronic acid loss of the cartilage matrix. Investigation of the molecular mechanisms in human articular chondrocytes showed the anti-inflammatory effect of purple corn anthocyanins and the metabolite, protocatechuic acid (PCA) on AGEs induced human articular chondrocytes via inactivation of the NFκb and MAPK signaling pathways. This finding suggests that purple corn anthocyanins and PCA may help ameliorate AGEs mediated inflammation and diabetes-mediated cartilage degradation.

Efficient engraftment of genetically modified cells is necessary to ameliorate central nervous system involvement of murine model of mucopolysaccharidosis type II by hematopoietic stem cell targeted gene therapy

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disease (LSD) caused by a deficiency of the iduronate-2-sulfatase (IDS) that catabolizes glycosaminoglycans (GAGs). Abnormal accumulations of GAGs in somatic cells lead to various manifestations including central nervous system (CNS) disease. Enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT) are the currently available therapy for MPS II, but both therapies fail to improve CNS manifestations. We previously showed that hematopoietic stem cell targeted gene therapy (HSC-GT) with lethal irradiation improved CNS involvement in a murine model of MPS II which lacks the gene coding for IDS. However, the strong preconditioning, with lethal irradiation, would cause a high rate of morbidity and mortality. Therefore, we tested milder preconditioning procedures with either low dose irradiation or low dose irradiation plus an anti c-kit monoclonal antibody (ACK2) to assess CNS effects in mice with MPS II after HSC-GT. Mice from all the HSC-GT groups displayed super-physiological levels of IDS enzyme activity and robust reduction of abnormally accumulated GAGs to the wild type mice levels in peripheral organs. However, only the mice treated with lethal irradiation showed significant cognitive function improvement as well as IDS elevation and GAG reduction in the brain.These results suggest that an efficient engraftment of genetically modified cells for HSC-GT requires strong preconditioning to ameliorate CNS involvement in cases with MPS II.

The long-term safety and efficacy of vestronidase alfa, rhGUS enzyme replacement therapy, in subjects with mucopolysaccharidosis VII

Vestronidase alfa (recombinant human beta-glucuronidase) is an enzyme replacement therapy (ERT) for Mucopolysaccharidosis (MPS) VII, a highly heterogeneous, ultra-rare disease. Twelve subjects, ages 8–25 years, completed a Phase 3, randomized, placebo-controlled, blind-start, single crossover study (UX003-CL301; NCT02377921), receiving 24–48 weeks of vestronidase alfa 4 mg/kg IV. All 12 subjects completed the blind-start study, which showed significantly reduced urinary glycosaminoglycans (GAG) and clinical improvement in a multi-domain responder index, and enrolled in a long-term, open-label, extension study (UX003-CL202; NCT02432144). Here, we report the final results of the extension study, up to an additional 144 weeks after completion of the blind-start study.Three subjects (25%) completed all 144 weeks of study, eight subjects (67%) ended study participation before Week 144 to switch to commercially available vestronidase alfa, and one subject discontinued due to non-compliance after receiving one infusion of vestronidase alfa in the extension study. The safety profile of vestronidase alfa in the extension study was consistent with observations in the preceding blind-start study, with most adverse events mild to moderate in severity. There were no treatment or study discontinuations due to AEs and no noteworthy changes in a standard safety chemistry panel. Out of the eleven subjects who tested positive for anti-drug antibodies at any time during the blind-start or extension study, including the baseline assessment in the blind-start study, seven subjects tested positive for neutralizing antibodies and all seven continued to demonstrate a reduction in urinary GAG levels. There was no association between antibody formation and infusion associated reactions.Subjects receiving continuous vestronidase alfa treatment showed a sustained urinary GAG reduction and clinical response evaluated using a multi-domain responder index that includes assessments in pulmonary function, motor function, range of motion, mobility, and visual acuity. Reduction in fatigue was also maintained in the overall population. As ERT is not expected to cross the blood brain barrier, limiting the impact on neurological signs of disease, and not all subjects presented with neurological symptoms, outcomes related to central nervous system pathology are not focused on in this report.Results from this study show the long-term safety and durability of clinical efficacy in subjects with MPS VII with long-term vestronidase alfa treatment.

Neonatal nonviral gene editing with the CRISPR/Cas9 system improves some cardiovascular, respiratory, and bone disease features of the mucopolysaccharidosis I phenotype in mice.

Mucopolysaccharidosis type I (MPS I) is caused by deficiency of alpha-L-iduronidase (IDUA), leading to multisystemic accumulation of glycosaminoglycans (GAG). Untreated MPS Ipatients maydie in the first decades of life, mostly due to cardiovascular and respiratory complications. We previously reported that the treatment of newborn MPS I mice with intravenous administration of lipossomal CRISPR/Cas9 complexes carrying the murine Idua geneaiming at the ROSA26 locus resulted in long-lasting IDUA activity and GAG reduction in varioustissues. Following this, the present study reports the effects of gene editing in cardiovascular, respiratory, bone, and neurologic functions in MPS I mice. Bone morphology, specifically the width of zygomatic and femoral bones, showed partial improvement. Although heart valves were still thickened, cardiac mass and aortic elastin breaks were reduced, with normalization of aortic diameter. Pulmonary resistance was normalized, suggesting improvement in respiratory function. In contrast, behavioral abnormalities and neuroinflammation still persisted, suggesting deterioration of the neurological functions. The set of results shows that gene editing performed in newborn animals improved some manifestations of the MPS I disorder in bone, respiratory, and cardiovascular systems. However, further studies will be imperative to find better delivery strategies to reach "hard-to-treat" tissues to ensure better systemic and neurological effects.

Extensive Metabolic Correction of Hurler Disease By Hematopoietic Stem Cell-Based Gene Therapy: Preliminary Results from a Phase I/II Trial

Extensive Metabolic Correction of Hurler Disease By Hematopoietic Stem Cell-Based Gene Therapy: Preliminary Results from a Phase I/II Trial

High Dose Hematopoietic Stem Cell Transplantation Leads to Rapid Hematopoietic and Microglia Recovery and Disease Correction in a Mouse Model of Hurler Syndrome

Background . Allogeneic hematopoietic stem cell transplant (HSCT) is a promising approach to halt disease progression and prevent or ameliorate neurological symptoms arising from select inherited metabolic disorders (IMDs). Donor-derived cells, including microglia, limit disease progression post-HSCT via production of normal enzyme in a process called cross-correction. A standard cell dose used in HSCT is sub-optimal, resulting in delayed hematopoietic recovery and slower correction of central nervous system (CNS) defects (Lund et al BBMT 2019). To address these limitations, we developed MGTA-456, a cell therapy that contains large numbers of CD34+ cells and has led to accelerated neutrophil recovery and 100% engraftment post-HSCT in patients with malignant and non-malignant diseases (Wagner et al Blood 2017; Orchard et al AAN 2019). We previously showed that MGTA-456 leads to faster hematopoietic and microglia recovery in the brains of NSG mice (Goncalves et al AAN 2019); however, the impact of cell dose on disease outcomes and mechanism of cross-correction are unknown. Here, we show that faster and greater hematopoietic and microglia recovery leads to rapid and complete resolution of disease endpoints in a mouse model of mucopolysaccharidosis I (Hurler syndrome) and that, mechanistically, donor engraftment in the brain is required for disease cross-correction. Results . To determine whether cell dose impacts microglial engraftment, CD45.2 mice were conditioned with a clinically-relevant, myeloablative dose of busulfan and transplanted with increasing doses of CD45.1 bone marrow cells, beginning with 0.3x106 cells/mouse (2x106 cells/kg) based on allometric scaling to model high dose cell therapies. A dose-dependent increase in microglia was observed as early as 1 week post-HSCT, where 10x106 cells led to a 26-fold higher number of donor microglia compared to 0.3x106 cells (p<0.01), an effect that was sustained through 16 weeks post-HSCT (p<0.001). Despite high donor chimerism in the periphery at all cell doses (75-99%), only partial chimerism was observed in the brain. At 16 weeks, donor microglia represented only 2% of microglia after transplant of 0.3x106 cells but this was increased to 35% of total microglia in the brain following transplant of 10x106 cells. These data indicate that while busulfan can facilitate a low level of microglia engraftment, this effect can be enhanced by transplant of high cell doses. To evaluate the impact of cell dose on disease outcomes, we transplanted a low (0.3x106) or high (10x106) cell dose of wild-type bone marrow cells into busulfan-conditioned Idua-/- mice, a model of Hurler syndrome. At 1 month post-HSCT, peripheral donor myeloid chimerism was >75% and >99% for 0.3x106 and 10x106 cells, respectively. In the brain, transplant of 10x106 cells led to significantly higher donor microglial engraftment versus 0.3x106 cells (Figure A). Notably, high cell dose resulted in significantly higher levels of IDUA enzyme in the brain (Figure B), reduced levels of β-hexosaminidase and glycosaminoglycan (GAG) substrate, and normalization of behavioral outcomes, including rotarod performance, to wild type levels (Figure C). In peripheral tissues, transplant of 10x106 cells, but not 0.3x106 cells, led to a reduction of GAGs to wild type levels as early as 1 week post-HSCT (p<0.01). To determine if donor engraftment in the brain is required for cross-correction, we transplanted 10x106 cells into mice conditioned with a myeloablative dose of treosulfan, which is not sufficient to condition the brain for microglia engraftment. Treosulfan conditioning, followed by high dose HSCT, led to >99% donor myeloid chimerism in the periphery but neither increased microglial levels nor corrected CNS defects (Figures A-C), suggesting that donor engraftment in the brain is required for disease modification. Long-term outcomes and impact on skeletal phenotype in this model will also be presented. Conclusions . We demonstrate that high dose HSCT leads to robust microglia engraftment in the brain and improved disease endpoints. These data suggest that strategies to increase cell dose, such as MGTA-456, may accelerate resolution of neurologic disease in patients with IMDs. Similar approaches, possibly coupled with gene modification technologies, could be used to improve microglial function in other neurodegenerative diseases where defective microglia have been implicated. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Brooks:Magenta Therapeutics: Employment, Equity Ownership. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Spatiotemporal Expression of 3-B-3(-) and 7-D-4 Chondroitin Sulfation, Tissue Remodeling, and Attempted Repair in an Ovine Model of Intervertebral Disc Degeneration.

Examination of intervertebral disc (IVD) regeneration in an ovine annular lesion model. Sulfation motifs are important functional determinants in glycosaminoglycans (GAGs). Previous studies have correlated 3-B-3(-) and 7-D-4 chondroitin sulfate (CS) motifs in tissues undergoing morphogenetic transition in development. We hypothesize that these motifs may also be expressed in degenerate IVDs and may represent a reparative response. Induction of disc degeneration by 5 mm or 6 × 20 mm lesions in the annulus fibrosus (AF) over 6 or 3 to 6 months postoperation (PO). Tissue sections were stained with toluidine blue-fast green, 3-B-3(-) and 7-D-4 CS-sulfation motifs were immunolocalized in 3-month PO 6 × 20 mm lesion IVDs. Sulfated glycosaminoglycan (GAG), 3-B-3(-), and 7-D-4 epitopes were quantitated by ELISIA (enzyme-linked immunosorbent inhibition assay) in extracts of AF (lesion site and contralateral half) and nucleus pulposus (NP) 0, 3, and 6 months PO. Collagenous overgrowth of lesions occurred in the outer AF. Chondroid metaplasia in ~20% of the 6 × 20 mm affected discs resulted in integration of an outgrowth of NP tissue with the inner AF lamellae preventing propagation of the lesion. 3-B-3(-) and 7-D-4 CS sulfation motifs were immunolocalized in this chondroid tissue. ELISIA quantified CS sulfation motifs demonstrating an increase 3 to 6 months PO in the AF lesion and a reduction in sulfated GAG not evident in the contralateral AF. (1) Outer annular lesions underwent spontaneous repair. (2) Chondroid metaplasia of the inner 6 × 20 mm defect prevented its propagation suggesting an apparent reparative response.

Long-term outcomes of enzyme replacement therapy for Taiwanese patients with Mucopolysaccharidosis I

Long-term outcomes of enzyme replacement therapy for Taiwanese patients with Mucopolysaccharidosis I

Dose-Dependent Effect of Mesenchymal Stromal Cell Recruiting Chemokine CCL25 on Porcine Tissue-Engineered Healthy and Osteoarthritic Cartilage.

Thymus-expressed chemokine (CCL25) is a potent cell attractant for mesenchymal stromal cells, and therefore it is a candidate for in situ cartilage repair approaches focusing on the recruitment of endogenous repair cells. However, the influence of CCL25 on cartilage is unknown. Accordingly, in this study, we investigated the effect of CCL25 on tissue-engineered healthy and osteoarthritic cartilage. Porcine chondrocytes were cultured in a three-dimensional (3D) micromass model that has been proven to mimic key-aspects of human cartilage and osteoarthritic alterations upon stimulation with tumor necrosis factor-α (TNF-α). Micromass cultures were stimulated with CCL25 (0, 0.05, 0.5, 5, 50, 500 nmol/L) alone or in combination with 0.6 nmol/L TNF-α for seven days. Effects were evaluated by life/dead staining, safranin O staining, histomorphometrical analysis of glycosaminoglycans (GAGs), collagen type II (COL2A1) real-time RT-PCR and Porcine Genome Array analysis. 500 nmol/L CCL25 led to a significant reduction of GAGs and COL2A1 expression and induced the expression of matrix metallopeptidases (MMP) 1, MMP3, early growth response protein 1 (EGR1), and superoxide dismutase 2 (SOD2). In concentrations lower than 500 nmol/L, CCL25 seems to be a candidate for in situ cartilage repair therapy approaches.

An Alternative Solution to Functional Neuroanatomy: The CLAIR Atlas

Nonsense suppression therapy is a therapeutic approach aimed at treating genetic diseases caused by in-frame premature termination codons (PTCs; also commonly known as nonsense mutations). This approach utilizes compounds that suppress translation termination at PTCs, which allows translation to continue and partial levels of deficient protein function to be restored. We hypothesize that suppression therapy can attenuate the lysosomal storage disease mucopolysaccharidosis type I-Hurler (MPS I-H), the severe form of α-l-iduronidase deficiency. α-l-iduronidase participates in glycosaminoglycan (GAG) catabolism and its insufficiency causes progressive GAG accumulation and onset of the MPS I-H phenotype, which consists of multiple somatic and neurological defects. 60–80% of MPS I-H patients carry a nonsense mutation in the IDUA gene. We previously showed that 2-week treatment with the designer aminoglycoside NB84 restored enough α-l-iduronidase function via PTC suppression to reduce tissue GAG accumulation in the Iduatm1Kmke MPS I-H mouse model, which carries a PTC homologous to the human IDUA-W402X nonsense mutation. Here we report that long-term NB84 administration maintains α-l-iduronidase activity and GAG reduction in Iduatm1Kmke mice throughout a 28-week treatment period. An examination of more complex MPS I-H phenotypes in Iduatm1Kmke mice following 28-week NB84 treatment revealed significant moderation of the disease in multiple tissues, including the brain, heart and bone, that are resistant to current MPS I-H therapies. This study represents the first demonstration that long-term nonsense suppression therapy can moderate progression of a genetic disease.

Targeting Root Cause by Systemic scAAV9-hIDS Gene Delivery: Functional Correction and Reversal of Severe MPS II in Mice

No treatment is available to address the neurological need and reversibility of MPS II. We developed a scAAV9-hIDS vector to deliver the human iduronate-2-sulfatase gene and test it in mouse model. We treated MPS II mice at different disease stages with an intravenous injection of scAAV9-mCMV-hIDS at different doses. The treatments led to rapid and persistent restoration of IDS activity and the reduction of glycosaminoglycans (GAG) throughout the CNS and somatic tissues in all cohorts. Importantly, the vector treatment at up to age 6 months improved behavior performance in the Morris water maze and normalized the survival. Notably, vector treatment at age 9 months also resulted in persistent rIDS expression and GAG clearance in MPS II mice, and the majority of these animals survived within the normal range of lifespan. Notably, the vector delivery did not result in any observable adverse events or detectable systemic toxicity in any treated animal groups. We believe that we have developed a safe and effective gene therapy for treating MPS II, which led to recent IND approval for a phase 1/2 clinical trial in MPS II patients, further supporting the extended potential of the demonstrated systemic rAAV9 gene delivery platform for broad disease targets.

AGE-RELATED DETERIORATION OF BONE TOUGHNESS IS RELATED TO DIMINISHING AMOUNT OF MATRIX GLYCOSAMINOGLYCANS (GAGS).

ABSTRACTHydration status significantly affects the toughness of bone. In addition to the collagen phase, recent evidence shows that glycosaminoglycans (GAGs) of proteoglycans (PGs) in the extracellular matrix also play a pivotal role in regulating the tissue‐level hydration status of bone, thereby affecting the tissue‐level toughness of bone. In this study, we hypothesized that the amount of GAGs in bone matrix decreased with age and such changes would lead to reduction in bound water and subsequently result in a decrease in the tissue‐level toughness of bone. To test the hypothesis, nanoscratch tests were conducted to measure the tissue‐level toughness of human cadaveric bone specimens, which were procured only from male donors in three different age groups: young (aged 26 ± 6 years), mid‐aged (aged 52 ± 5 years), and elderly (aged 73 ± 5 years), with 6 donors in each group. Biochemical and histochemical assays were performed to determine the amount and major subtypes of GAGs and proteoglycans in bone matrix. In addition, low‐field nuclear magnetic resonance (NMR) measurements were implemented to determine bound water content in bone matrix. The results demonstrated that aging resulted in a statistically significant reduction (17%) of GAGs in bone matrix. Concurrently, a significant deterioration (20%) of tissue‐level toughness of bone with age was observed. Most importantly, the deteriorated tissue‐level toughness of bone was associated significantly with the age‐related reduction (40%) of bound water, which was partially induced by the decrease of GAGs in bone matrix. Furthermore, we identified that chondroitin sulfate (CS) was a major subtype of GAGs, and the amount of CS decreased with aging accompanied with a decrease of biglycan that is a major subtype of PGs in bone. The findings of this study suggest that reduction of GAGs in bone matrix is likely one of the molecular origins for age‐related deterioration of bone quality. © 2017 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Postnatal lethality and chondrodysplasia in mice lacking both chondroitin sulfate N-acetylgalactosaminyltransferase-1 and -2.

Chondroitin sulfate (CS) is a sulfated glycosaminoglycan (GAG) chain. In cartilage, CS plays important roles as the main component of the extracellular matrix (ECM), existing as side chains of the major cartilage proteoglycan, aggrecan. Six glycosyltransferases are known to coordinately synthesize the backbone structure of CS; however, their in vivo synthetic mechanism remains unknown. Previous studies have suggested that two glycosyltransferases, Csgalnact1 (t1) and Csgalnact2 (t2), are critical for initiation of CS synthesis in vitro. Indeed, t1 single knockout mice (t1 KO) exhibit slight dwarfism and a reduction in CS content in cartilage compared with wild-type (WT) mice. To reveal the synergetic roles of t1 and t2 in CS synthesis in vivo, we generated systemic single and double knockout (DKO) mice and cartilage-specific t1 and t2 double knockout (Col2-DKO) mice. DKO mice exhibited postnatal lethality, whereas t2 KO mice showed normal size and skeletal development. Col2-DKO mice survived to adulthood and showed severe dwarfism compared with t1 KO mice. Histological analysis of epiphyseal cartilage from Col2-DKO mice revealed disrupted endochondral ossification, characterized by drastic GAG reduction in the ECM. Moreover, DKO cartilage had reduced chondrocyte proliferation and an increased number of apoptotic chondrocytes compared with WT cartilage. Conversely, primary chondrocyte cultures from Col2-DKO knee cartilage had the same proliferation rate as WT chondrocytes and low GAG expression levels, indicating that the chondrocytes themselves had an intact proliferative ability. Quantitative RT-PCR analysis of E18.5 cartilage showed that the expression levels of Col2a1 and Ptch1 transcripts tended to decrease in DKO compared with those in WT mice. The CS content in DKO cartilage was decreased compared with that in t1 KO cartilage but was not completely absent. These results suggest that aberrant ECM caused by CS reduction disrupted endochondral ossification. Overall, we propose that both t1 and t2 are necessary for CS synthesis and normal chondrocyte differentiation but are not sufficient for all CS synthesis in cartilage.

RTB lectin-mediated delivery of lysosomal α-l-iduronidase mitigates disease manifestations systemically including the central nervous system.

Mucopolysaccharidosis type I (MPS I) is a lysosomal disease resulting from deficiency in the α-L-iduronidase (IDUA) hydrolase and subsequent accumulation of glycosaminoglycan (GAG). Clinically, enzyme replacement therapy (ERT) with IDUA achieves negligible neurological benefits presumably due to blood-brain-barrier (BBB) limitations. To investigate the plant lectin ricin B chain (RTB) as a novel carrier for enzyme delivery to the brain, an IDUA:RTB fusion protein (IDUAL), produced in N. benthamiana leaves, was tested in a murine model of Hurler syndrome (MPS I). Affect mice (n=3 for each group) were intravenously injected with a single dose of IDUAL (0.58, 2 or 5.8mgIDUAequivalents/kg) and analyzed after 24h. IDUA activities in liver, kidney and spleen increased significantly, and liver GAG levels were significantly reduced in all three groups. Plasma IDUA levels for all treated groups were high at 1h after injection and decreased by 95% at 4h, indicating efficient distribution into tissues. For long-term evaluations, IDUAL (0.58 or 2mg/kg, 8 weekly injections) was intravenously injected into MPS I mice (n=12 for each group). Thirteen days after the 8th injection, significant IDUA activity was detected in the liver and spleen. GAG levels in tissues including the brain cortex and cerebellum were significantly reduced in treated animals. Treated MPS I mice also showed significant improvement in neurocognitive testing. ELISA results showed that while there was a significant antibody response against IDUAL and plant-derived IDUA, there was no significant antibody response to RTB. No major toxicity or adverse events were observed. Together, these results showed that infusion of IDUAL allowed for significant IDUA levels and GAG reduction in the brain and subsequent neurological benefits. This RTB-mediated delivery may have significant implications for therapeutic protein delivery impacting a broad spectrum of lysosomal, and potentially neurological diseases.

Elements of lentiviral vector design toward gene therapy for treating mucopolysaccharidosis I

Mucopolysaccharidosis type I (MPS I) is a lysosomal disease caused by α-l-iduronidase (IDUA) deficiency and accumulation of glycosaminoglycans (GAG). Lentiviral vector encoding correct IDUA cDNA could be used for treating MPS I. To optimize the lentiviral vector design, 9 constructs were designed by combinations of various promoters, enhancers, and codon optimization. After in vitro transfection into 293FT cells, 5 constructs achieved the highest IDUA activities (5613 to 7358nmol/h/mg protein). These 5 candidate vectors were then tested by injection (1×107TU/g) into neonatal MPS I mice. After 30days, one vector, CCEoIDW, achieved the highest IDUA levels: 2.6% of wildtype levels in the brain, 9.9% in the heart, 200% in the liver and 257% in the spleen. CCEoIDW achieved the most significant GAG reduction: down 49% in the brain, 98% in the heart, 100% in the liver and 95% in the spleen. Further, CCEoIDW had the lowest transgene frequency, especially in the gonads (0.03±0.01 copies/100 cells), reducing the risk of insertional mutagenesis and germ-line transmission. Therefore, CCEoIDW is selected as the optimal lentiviral vector for treating MPS I disease and will be applied in large animal preclinical studies. Further, taken both in vitro and in vivo comparisons together, codon optimization, use of EF-1α promoter and woodchuck hepatitis virus posttranscriptional response element (WPRE) could enhance transgene expression. These results provided a better understanding of factors contributing efficient transgene expression in lentiviral gene therapies.

484. In Vivo Zinc-Finger Nuclease Mediated Iduronate-2-Sulfatase (IDS) Target Gene Insertion and Correction of Metabolic Disease in a Mouse Model of Mucopolysaccharidosis Type II (MPS II)

Hunter syndrome (Mucopolysaccharidosis Type II, MPS II) is a rare X-linked lysosomal disorder caused by lack of functional iduronate-2 sulfatase (IDS) enzyme and subsequent accumulation of glycosaminoglycans (GAG) in affected individuals. Manifestations include skeleton dysplasia, splenohepatomegaly, cardiopulmonary obstruction, and shortened life expectancy. In severe cases there is also neurologic impairment. Enzyme replacement therapy (ERT) is currently the only FDA-approved treatment to manage disease progression; however, ERT does not affect neurological aspects of the disease and requires that patients receive long and costly infusions of replacement factor on a frequent basis. We have developed a zinc-finger nuclease (ZFN) approach to insert the human IDS (hIDS) coding sequence into the albumin locus using AAV2/8 vectors. In this study IDS-deficient MPS II mice (n= 8-13 per group, age 7-8 weeks) were treated by intravenous infusion of a mixture of ZFN-encoding AAV vectors along with an AAV vector encoding the hIDS partial cDNA flanked by albumin sequence homology arms at three different vector doses. Wild-type littermates, untreated MPS II mice, and MPS II animals infused only with the hIDS donor vector (without ZFN-encoding vectors) were included as controls. Successful insertion of the hIDS coding sequence will result in hIDS expression regulated by the endogenous albumin promoter. Plasma and tissue IDS activities as well as urine and tissue GAG contents were monitored throughout the study to evaluate the effectiveness of the treatment. Sufficient animals were maintained for neurobehavioral testing at four-months post-injection to determine whether the treatment is neurologically beneficial. We found that IDS activities in the plasma of the treated groups were 10- to 100-fold higher than wild-type and stably expressed through the entire study duration in a dose-dependent fashion, while only very low levels of IDS activity were found in the animals infused with hIDS donor vector alone. At 4 weeks post-treatment IDS activities in peripheral tissues ranged from 1% to 200% wild-type in a dose-dependent fashion, while in the hIDS donor-only group enzyme activity was not detected in any tissue except liver (10% that of wild-type). We observed up to 2% of the wild-type IDS activity in the brains of animals administered the complete set of AAV vectors, while no IDS activity was observed in the brains of animals infused with the IDS donor vector alone. Urine GAGs were reduced in all of the ZFN + Donor treatment groups regardless of the vector dose. Tissue GAGs in the treatment groups were also decreased, but GAG content in the brain was not different from untreated MPS II litter mates at the initial analysis conducted four weeks post-treatment. No tissue GAG reduction was observed in animals infused with hIDS donor vector alone. Before conclusion of this study, animals from all six groups will be tested in the Barnes Maze as neurobehavioral assessment to determine the neurological effect of targeted hIDS expression in the liver. These results together with hIDS expression and GAG level data from final necropsy tissues will be presented. These results demonstrate that ZFN can be effectively used to mediate in vivo insertion of the hIDS coding sequence into the albumin locus with resultant stable and high-level hIDS enzyme expression and metabolic correction in MPS II.