Discovery of allosteric regulators with clinical potential to stabilize alpha-L-iduronidase in mucopolysaccharidosis type I.

Mucopolysaccharidosis type I (MPS I) is an inherited lysosomal disease caused by lowered activity of the enzyme alpha-L-iduronidase (IDUA). Current therapeutic options show limited efficacy and do not treat some important aspects of the disease. Therefore, it may be advantageous to identify strategies that could improve the efficacy of existing treatments. Pharmacological chaperones are small molecules that protect proteins from degradation, and their use in combination with enzyme replacement therapy (ERT) has been proposed as an alternative therapeutic strategy. Using the SEE-Tx® proprietary computational drug discovery platform, a new allosteric ligand binding cavity in IDUA was identified distal from the active site. Virtual high-throughput screening of approximately 5 million compounds using the SEE-Tx® docking platform identified a subset of small molecules that bound to the druggable cavity and functioned as novel allosteric chaperones of IDUA. Experimental validation by differential scanning fluorimetry showed an overall hit rate of 11.4%. Biophysical studies showed that one exemplary hit molecule GT-01803 bound to (Kd = 22 µM) and stabilized recombinant human IDUA (rhIDUA) in a dose-dependent manner. Co-administration of rhIDUA and GT-01803 increased IDUA activity in patient-derived fibroblasts. Preliminary in vivo studies have shown that GT-01803 improved the pharmacokinetic (PK) profile of rhIDUA, increasing plasma levels in a dose-dependent manner. Furthermore, GT-01803 also increased IDUA enzymatic activity in bone marrow tissue, which benefits least from standard ERT. Oral bioavailability of GT-01803 was found to be good (50%). Overall, the discovery and validation of a novel allosteric chaperone for rhIDUA presents a promising strategy to enhance the efficacy of existing treatments for MPS I. The compound's ability to increase rhIDUA activity in patient-derived fibroblasts and its good oral bioavailability underscore its potential as a potent adjunct to ERT, particularly for addressing aspects of the disease less responsive to standard treatment.

Modeling skeletal dysplasia in Hurler syndrome using patient-derived bone marrow osteoprogenitor cells.

Dysostosis multiplex is a major cause of morbidity in Hurler syndrome (mucopolysaccharidosis type IH [MPS IH], OMIM #607014) because currently available therapies have limited success in its prevention and reversion. Unfortunately, the elucidation of skeletal pathogenesis in MPS IH is limited by difficulties in obtaining bone specimens from pediatric patients and poor reproducibility in animal models. Thus, the application of experimental systems that can be used to dissect cellular and molecular mechanisms underlying the skeletal phenotype of MPS IH patients and to identify effective therapies is highly needed. Here, we adopted in vitro/in vivo systems based on patient-derived bone marrow stromal cells to generate cartilaginous pellets and bone rudiments. Interestingly, we observed that heparan sulphate accumulation compromised the remodeling of MPS IH cartilage into other skeletal tissues and other critical aspects of the endochondral ossification process. We also noticed that MPS IH hypertrophic cartilage was characterized by dysregulation of signaling pathways controlling cartilage hypertrophy and fate, extracellular matrix organization, and glycosaminoglycan metabolism. Our study demonstrates that the cartilaginous pellet-based system is a valuable tool to study MPS IH dysostosis and to develop new therapeutic approaches for this hard-to-treat aspect of the disease. Finally, our approach may be applied for modeling other genetic skeletal disorders.

α-L-iduronidase fused with humanized anti-human transferrin receptor antibody (lepunafusp alfa) for mucopolysaccharidosis type I: A phase 1/2 trial.

Mucopolysaccharidosis type I (MPS I) causes systemic accumulation of glycosaminoglycans due to a genetic deficiency of α-L-iduronidase (IDUA), which results in progressive systemic symptoms affecting multiple organs, including the central nervous system (CNS). Because the blood-brain barrier (BBB) prevents enzymes from reaching the brain, enzyme replacement therapy is effective only against the somatic symptoms. Hematopoietic stem cell transplantation can address the CNS symptoms, but the risk of complications limits its applicability. We have developed a novel genetically modified protein consisting of IDUA fused with humanized anti-human transferrin receptor antibody (lepunafusp alfa; JR-171), which has been shown in nonclinical studies to be distributed to major organs, including the brain, bringing about systemic reductions in heparan sulfate (HS) and dermatan sulfate concentrations. Subsequently, a first-in-human study was conducted to evaluate the safety, pharmacokinetics, and exploratory efficacy of JR-171 in 18 patients with MPS I. No notable safety issues were observed. Plasma drug concentration increased dose dependently and reached its maximum approximately 4 h after the end of drug administration. Decreased HS in the cerebrospinal fluid suggested successful delivery of JR-171 across the BBB, while suppressed urine and serum concentrations of the substrates indicated that its somatic efficacy was comparable to that of laronidase.

First Three Years' Experience of Mucopolysaccharidosis Type-I Newborn Screening in California.

OBJECTIVE: To report on the first 3 years of mucopolysaccharidosis type I (MPS I) newborn screening (NBS) in the large and diverse state of California. STUDY DESIGN: The California Genetic Disease Screening Program began universal NBS for MPS I on August 29, 2018. The screening uses a 2-tiered approach: an α-L-iduronidase (IDUA) enzyme activity assay followed by DNA sequencing for variants in the IDUA gene. RESULTS: As of August 29, 2021, 1 295 515 California newborns were screened for MPS I. In tier 1 of screening, 329 (0.025%) had an IDUA enzyme measurement below the cutoff and underwent tier-2 IDUA DNA sequencing. After tier 2, 146 (0.011%) newborns were screen positive, all of whom were referred to a metabolic Special Care Center for follow-up. After long-term follow-up, 7 cases were resolved as severe MPS I (Hurler syndrome) and 2 cases as attenuated MPS I for an MPS I birth prevalence of 1/143 946. DNA sequencing identified 107 unique IDUA variants among a total of 524 variants; 65% were known pseudodeficiency alleles, 25% were variants of uncertain significance, and 10% were pathogenic variants. CONCLUSIONS: As a result of a 2-tiered NBS approach, 7 newborns diagnosed with Hurler syndrome had received early treatment for MPS I. Continuation of California's long-term follow-up program will be crucial for further understanding the complex genotype-phenotype relationships of MPS I.

Phenotypic characterisation of the Mucopolysaccharidosis Type I (MPSI) Idua-W392X mouse model reveals increased anxiety-related traits in female mice.

Mucopolysaccharidosis Type I (MPSI) is a rare inherited lysosomal storage disease that arises due to mutations in the IDUA gene. Defective alpha-L-iduronidase (IDUA) enzyme is unable to break down glucosaminoglycans (GAGs) within the lysosomes and, as a result, there is systemic accumulation of undegraded products in lysosomes throughout the body leading to multi-system disease. Here, we characterised the skeletal/craniofacial, neuromuscular and behavioural outcomes of the MPSI Idua-W392X mouse model. We demonstrate that Idua-W392X mice have gross craniofacial abnormalities, showed signs of kyphosis, and show signs of hypoactivity compared to wild-type mice. X-ray imaging analysis revealed significantly shorter and wider tibias and femurs, significantly wider snouts, increased skull width and significantly thicker zygomatic arch bones in Idua-W392X female mice compared to wild-type mice at 9 and 10.5 months of age. Idua-W392X mice display decreased muscle strength, especially in the forelimbs, which is already apparent from 3 months of age. Female Idua-W392X mice display hypoactivity in the open-field test from 9 months of age and anxiety-like behaviour at 10 months of age. As these behaviours have been identified in Hurler children, the MPSI Idua-W392X mouse model may be important for the investigation of new therapeutic approaches for MPSI-Hurler.

Identification of an α-l-iduronidase (IDUA) M1T mutation in a Chinese family with autosomal recessive mucopolysaccharidosis I.

Mucopolysaccharidoses (MPS) are a group of rare congenital metabolic disorders caused by the deficiency or low activity of enzymes required for glycosaminoglycans degradation. Mutations in the α-l-iduronidase gene (IDUA) are associated with mucopolysaccharidosis type I (MPS I). Our study here aims to identify an MPS-related gene mutation in a typical patient with MPS and to further explore the possible pathogenic mechanism. We identified a homozygous c. 2T>C (p.M1T) change in IDUA as the pathogenic mutation in this individual (both parents were identified as carriers of the mutation), with IDUA enzyme activity significantly decreased. We further established an MPS I-related zebrafish model using IDUA-specific morpholino (MO) to suppress gene expression, and found that IDUA-MO zebrafish exhibited characteristic disease phenotypes with deficiency of IDUA. Transcriptome profiling of zebrafish larvae revealed 487 genes that were significantly altered when IDUA was depleted. TP53 signaling and LC3/GABARAP family protein-mediated autophagy were significantly upregulated in IDUA-MO zebrafish larvae. Moreover, leukotriene A4 hydrolase-mediated arachidonic acid metabolism was also upregulated. Introduction of wild-type human IDUA mRNA rescued developmental defects and aberrant signaling in IDUA-MO zebrafish larvae. In conclusion, our study provides potential therapeutic targets for the treatment of MPS I.

18-year follow-up of enzyme-replacement therapy in two siblings with attenuated mucopolysaccharidosis I.

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive disorder caused by the deficiency of α-L-iduronidase and characterized by a progressive course with multisystem involvement. Clinically, MPS I is divided into two forms: (1) severe (Hurler syndrome), which presents in infancy and is characterized by rapid progressive neurological involvement; (2) attenuated (Hurler/Scheie and Scheie syndromes), which displays a slower progression and absent to mild nervous system involvement. The specific treatment for attenuated MPS I consists of enzyme-replacement therapy with laronidase (human recombinant α-L-iduronidase, Aldurazyme). We present updated data after 18 years of laronidase treatment in two siblings affected by the attenuated form of MPS I who started therapy at 5 months and 5 years of age, respectively. Clinical and laboratory data of the siblings show that long-term enzyme replacement therapy may improve/stabilize many symptoms already present at the time of the diagnosis and reduce the disease progression. This study confirms that early diagnosis and early initiation of enzyme-replacement therapy are essential to modify positively the natural history of the attenuated form of MPS I.

Synthesis of Uronic Acid 1-Azasugars as Putative Inhibitors of α-Iduronidase, ß-Glucuronidase and Heparanase.

1-Azasugar analogues of l-iduronic acid (l-IdoA) and d-glucuronic acid (d-GlcA) and their corresponding enantiomers have been synthesized as potential pharmacological chaperones for mucopolysaccharidosis I (MPS I), a lysosomal storage disease caused by mutations in the gene encoding α-iduronidase (IDUA). The compounds were efficiently synthesized in nine or ten steps from d- or l-arabinose, and the structures were confirmed by X-ray crystallographic analysis of key intermediates. All compounds were inactive against IDUA, although l-IdoA-configured 8 moderately inhibited ß-glucuronidase (ß-GLU). The d-GlcA-configured 9 was a potent inhibitor of ß-GLU and a moderate inhibitor of the endo-ß-glucuronidase heparanase. Co-crystallization of 9 with heparanase revealed that the endocyclic nitrogen of 9 forms close interactions with both the catalytic acid and catalytic nucleophile.

Neurologic Recovery in MPS I and MPS II Mice by AAV9-Mediated Gene Transfer to the CNS After the Development of Cognitive Dysfunction.

The mucopolysaccharidoses (MPS) are a group of recessively inherited conditions caused by deficiency of lysosomal enzymes essential to the catabolism of glycosaminoglycans (GAG). MPS I is caused by deficiency of the lysosomal enzyme alpha-L-iduronidase (IDUA), while MPS II is caused by a lack of iduronate-2-sulfatase (IDS). Lack of these enzymes leads to early mortality and morbidity, often including neurological deficits. Enzyme replacement therapy has markedly improved the quality of life for MPS I and MPS II affected individuals but is not effective in addressing neurologic manifestations. For MPS I, hematopoietic stem cell transplant has shown effectiveness in mitigating the progression of neurologic disease when carried out in early in life, but neurologic function is not restored in patients transplanted later in life. For both MPS I and II, gene therapy has been shown to prevent neurologic deficits in affected mice when administered early, but the effectiveness of treatment after the onset of neurologic disease manifestations has not been characterized. To test if neurocognitive function can be recovered in older animals, human IDUA or IDS-encoding AAV9 vector was administered by intracerebroventricular injection into MPS I and MPS II mice, respectively, after the development of neurologic deficit. Vector sequences were distributed throughout the brains of treated animals, associated with high levels of enzyme activity and normalized GAG storage. Two months after vector infusion, treated mice exhibited spatial navigation and learning skills that were normalized, that is, indistinguishable from those of normal unaffected mice, and significantly improved compared to untreated, affected animals. We conclude that cognitive function was restored by AAV9-mediated, central nervous system (CNS)-directed gene transfer in the murine models of MPS I and MPS II, suggesting that gene transfer may result in neurodevelopment improvements in severe MPS I and MPS II when carried out after the onset of cognitive decline.

Effects of gentamicin inducing readthrough premature stop Codons: A study of alpha-L-iduronidase nonsense variants in COS-7 Cells.

Mucopolysaccharidosis type I Hurler syndrome (MPS IH) is a severe lysosomal storage disorder caused by alpha-l-iduronidase (IDUA) deficiency. Premature truncation mutations (PTC) are the most common (50%-70%) type of IDUA mutations and correlate with MPS IH. Nonsense suppression therapy is a therapeutic approach that aims to induce stop codon readthrough. The different ability of gentamicin to bind mutant mRNA in readthrough is determined by nucleotide sequence (PTC context: UGA > UAG > UAA) and inserted amino acid including the nucleotide position +4 of the PTC, as well as the mRNA secondary structure. We used COS-7 cells to investigate the functional characteristics of p.Q500X and p.R619X, IDUA variants and the effects of gentamicin in inducing stop codon readthrough of seven IDUA variants including p.Q500X, p.R619X, p.Q70X, p.E299X, p.W312X, p.Q380X, and p.W402X. Moreover, we performed prediction of RNA secondary structure using the online tool RNAfold. We found that cells treated with gentamicin showed significantly enhanced full-length IDUA expression and restored IDUA activity, in a dose-dependent manner, only in cells expressing cDNA with W312X, Q380X, W402X, and R619X. Among the readthrough-responsive variants, we observed UGA PTC in W312X, W402X and R619X; and UAG PTC with C at nucleotide +4 in Q380X. Changes of RNA secondary structure were noted only in mutants with readthrough-responsive variants including W312X, Q380X, W402X, and R619X. Additional preclinical studies of selected PTCs with potential readthrough, using drugs with less oto-nephrotoxicity, in patient's skin fibroblasts and animal model are necessary for the premise of personalized medicine.

MPSI Manifestations and Treatment Outcome: Skeletal Focus.

Mucopolysaccharidosis type I (MPSI) (OMIM #252800) is an autosomal recessive disorder caused by pathogenic variants in the IDUA gene encoding for the lysosomal alpha-L-iduronidase enzyme. The deficiency of this enzyme causes systemic accumulation of glycosaminoglycans (GAGs). Although disease manifestations are typically not apparent at birth, they can present early in life, are progressive, and include a wide spectrum of phenotypic findings. Among these, the storage of GAGs within the lysosomes disrupts cell function and metabolism in the cartilage, thus impairing normal bone development and ossification. Skeletal manifestations of MPSI are often refractory to treatment and severely affect patients' quality of life. This review discusses the pathological and molecular processes leading to impaired endochondral ossification in MPSI patients and the limitations of current therapeutic approaches. Understanding the underlying mechanisms responsible for the skeletal phenotype in MPSI patients is crucial, as it could lead to the development of new therapeutic strategies targeting the skeletal abnormalities of MPSI in the early stages of the disease.

Ataluren suppresses a premature termination codon in an MPS I-H mouse.

ABSTARCT: Suppressing translation termination at premature termination codons (PTCs), termed readthrough, is a potential therapy for genetic diseases caused by nonsense mutations. Ataluren is a compound that has shown promise for clinical use as a readthrough agent. However, some reports suggest that ataluren is ineffective at suppressing PTCs. To further evaluate the effectiveness of ataluren as a readthrough agent, we examined its ability to suppress PTCs in a variety of previously untested models. Using NanoLuc readthrough reporters expressed in two different cell types, we found that ataluren stimulated a significant level of readthrough. We also explored the ability of ataluren to suppress a nonsense mutation associated with Mucopolysaccharidosis I-Hurler (MPS I-H), a genetic disease that is caused by a deficiency of α-L-iduronidase that leads to lysosomal accumulation of glycosaminoglycans (GAGs). Using mouse embryonic fibroblasts (MEFs) derived from Idua-W402X mice, we found that ataluren partially rescued α-L-iduronidase function and significantly reduced GAG accumulation relative to controls. Two-week oral administration of ataluren to Idua-W402X mice led to significant GAG reductions in most tissues compared to controls. Together, these data reveal important details concerning the efficiency of ataluren as a readthrough agent and the mechanisms that govern its ability to suppress PTCs. KEY MESSAGES: Ataluren promotes readthrough of PTCs in a wide variety of contexts. Ataluren reduces glycosaminoglyan storage in MPS I-H cell and mouse models. Ataluren has a bell-shaped dose-response curve and a narrow effective range.

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

BACKGROUND: 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. METHODS: 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 6 months of age after performing behavioral tasks, and biochemical and molecular aspects were evaluated. RESULTS: 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. CONCLUSION: 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.

Hematopoietic Stem- and Progenitor-Cell Gene Therapy for Hurler Syndrome.

BACKGROUND: Allogeneic hematopoietic stem-cell transplantation is the standard of care for Hurler syndrome (mucopolysaccharidosis type I, Hurler variant [MPSIH]). However, this treatment is only partially curative and is associated with complications. METHODS: We are conducting an ongoing study involving eight children with MPSIH. At enrollment, the children lacked a suitable allogeneic donor and had a Developmental Quotient or Intelligence Quotient score above 70 (i.e., none had moderate or severe cognitive impairment). The children received autologous hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with an α-L-iduronidase (IDUA)-encoding lentiviral vector after myeloablative conditioning. Safety and correction of blood IDUA activity up to supraphysiologic levels were the primary end points. Clearance of lysosomal storage material as well as skeletal and neurophysiological development were assessed as secondary and exploratory end points. The planned duration of the study is 5 years. RESULTS: We now report interim results. The children's mean (±SD) age at the time of HSPC gene therapy was 1.9±0.5 years. At a median follow-up of 2.10 years, the procedure had a safety profile similar to that known for autologous hematopoietic stem-cell transplantation. All the patients showed prompt and sustained engraftment of gene-corrected cells and had supraphysiologic blood IDUA activity within a month, which was maintained up to the latest follow-up. Urinary glycosaminoglycan (GAG) excretion decreased steeply, reaching normal levels at 12 months in four of five patients who could be evaluated. Previously undetectable levels of IDUA activity in the cerebrospinal fluid became detectable after gene therapy and were associated with local clearance of GAGs. Patients showed stable cognitive performance, stable motor skills corresponding to continued motor development, improved or stable findings on magnetic resonance imaging of the brain and spine, reduced joint stiffness, and normal growth in line with World Health Organization growth charts. CONCLUSIONS: The delivery of HSPC gene therapy in patients with MPSIH resulted in extensive metabolic correction in peripheral tissues and the central nervous system. (Funded by Fondazione Telethon and others; ClinicalTrials.gov number, NCT03488394; EudraCT number, 2017-002430-23.).

Mucopolysaccharidoses type I gene therapy.

Mucopolysaccharidoses type I (MPS I) is an inherited metabolic disease characterized by a malfunction of the α-l-iduronidase (IDUA) enzyme leading to the storage of glycosaminoglycans in the lysosomes. This disease has longtime been studied as a therapeutic target for those studying gene therapy and many studies have been done using various vectors to deliver the IDUA gene for corrective treatment. Many vectors have difficulties with efficacy and insertional mutagenesis concerns including adeno-associated viral (AAV) vectors. Studies of AAV vectors treating MPS I have seemed promising, but recent deaths in gene therapy clinical trials for other inherited diseases using AAV vectors have left questions about their safety. Additionally, the recent modifications to adenoviral vectors leading them to target the vascular endothelium minimizing the risk of hepatotoxicity could lead to them being a viable option for MPS I gene therapy when coupled with gene editing technologies like CRISPR/Cas9.

STEP® vectors for rapid generation of stable transfected CHO cell pools and clones with high expression levels and product quality homogeneity of difficult-to-express proteins.

Many proteins produced in CHO cells need evaluation for their clinical and commercial potential. Traditional methods based on stable clone generation are slow and unsuitable for screening larger numbers of proteins, while transient expression technologies are fast but unpredictable regarding product quality and lacking an optional path to subcloning. The STEP® vector technology introduced here combines the best properties of both methods. STEP® vectors contain a strong transcriptional cassette driving expression of a bicistronic mRNA. The gene-of-interest (GOI) is cloned upstream of a functionally impaired zeocin resistance gene (FI-Zeo) whose translation is coupled to that of the GOI through an IRES. Stable transfected cells surviving zeocin selection produce high levels of FI-Zeo and thus, high levels of the GOI-encoded protein. By using different spacers, the translational coupling efficiency and selection strength can be controlled allowing maximization of expression of any GOI. Production of laronidase and factor VII (FVII) is presented as examples of unrelated, difficult-to-express (DTE) proteins. First step is rapid generation of transfected pools with the STEP® vectors. All high expressing surviving pools showed high product quality homogeneity as did monoclonal cell lines obtained from the top pools. Up to 500 µg/mL laronidase was obtained with virtually identical glycosylation profile as reference product. For FVII, cell specific productivity of 0.45 pg/cell/day with 50 IU/µg protein matched highest reported levels of reference product even before process development. Taken together, STEP® vector technology is ideally suited for rapid, small to large-scale production of DTE proteins compared to traditional methods.

Gene Therapy of Mucopolysaccharidosis Type I Mice: Repeated Administrations and Safety Assessment of pIDUA/Nanoemulsion Complexes.

BACKGROUND: Mucopolysaccharidosis type I (MPS I) is an inherited disorder caused by α-L-iduronidase (IDUA) deficiency. The available treatments are not effective in improving all signs and symptoms of the disease. OBJECTIVE: In the present study, we evaluated the transfection efficiency of repeated intravenous administrations of cationic nanoemulsions associated with the plasmid pIDUA (containing IDUA gene). METHODS: Cationic nanoemulsions were composed of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(amino[polyethylene glycol]- 2000) (DSPE-PEG), 1,2-dioleoyl-sn-glycero-3-trimethylammonium propane (DOTAP), medium- chain triglycerides, glycerol, and water and were prepared by high-pressure homogenization and were repeatedly administered to MPS I mice for IDUA production and gene expression. RESULTS: A significant increase in IDUA expression was observed in all organs analyzed, and IDUA activity tended to increase with repeated administrations when compared to our previous report when mice received a single administration of the same dose. In addition, GAGs were partially cleared from organs, as assessed through biochemical and histological analyzes. There was no presence of inflammatory infiltrate, necrosis, or signs of an increase in apoptosis. Furthermore, immunohistochemistry for CD68 showed a reduced presence of macrophage cells in treated than in untreated MPS I mice. CONCLUSION: These sets of results suggest that repeated administrations can improve transfection efficiency of cationic complexes without a significant increase in toxicity in the MPS I murine model.

Biomechanical and histological characterization of MPS I mice femurs.

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder characterized by alpha-L-iduronidase (IDUA) deficiency, an enzyme responsible for glycosaminoglycan degradation. Musculoskeletal impairment is an important component of the morbidity related to the disease, as it has a major impact on patients' quality of life. To understand how this disease affects bone structure, morphological, biomechanical and histological analyses of femurs from 3- and 6-month-old wild type (Idua +/+) and MPS I knockout mice (Idua -/-) were performed. Femurs from 3-month-old Idua -/- mice were found to be smaller and less resistant to fracture when compared to their age matched controls. In addition, at this age, the femurs presented important alterations in articular cartilage, trabecular bone architecture, and deposition of type I and III collagen. At 6 months of age, femurs from Idua -/- mice were more resistant to fracture than those from Idua +/+. Our results suggest that the abnormalities observed in bone matrix and articular cartilage in 3-month-old Idua -/- animals caused bone tissue to be less flexible and more likely to fracture, whereas in 6-month-old Idua -/- group the ability to withstand more load before fracturing than wild type animals is possibly due to changes in the bone matrix.

c.1898C>G/p.Ser633Trp Mutation in Alpha-L-Iduronidase: Clinical and Structural Implications.

Mucopolysaccharidosis type I is a rare autosomal recessive genetic disease caused by deficient activity of α-L-iduronidase. As a consequence of low or absent activity of this enzyme, glycosaminoglycans accumulate in the lysosomal compartments of multiple cell types throughout the body. Mucopolysaccharidosis type I has been classified into 3 clinical subtypes, ranging from a severe Hurler form to the more attenuated Hurler-Scheie and Scheie phenotypes. Over 200 gene variants causing the various forms of mucopolysaccharidosis type I have been reported. DNA isolated from dried blood spot was used to sequencing of all exons of the IDUA gene from a patient with a clinical phenotype of severe mucopolysaccharidosis type I syndrome. Enzyme activity of α-L-iduronidase was quantified by fluorimetric assay. Additionally, a molecular dynamics simulation approach was used to determine the effect of the Ser633Trp mutation on the structure and dynamics of the α-L-iduronidase. The DNA sequencing analysis and enzymatic activity shows a c.1898C>G mutation associated a patient with a homozygous state and α-L-iduronidase activity of 0.24 µmol/L/h, respectively. The molecular dynamics simulation analysis shows that the p.Ser633Trp mutation on the α-L-iduronidase affect significant the temporal and spatial properties of the different structural loops, the N-glycan attached to Asn372 and amino acid residues around the catalytic site of this enzyme. Low enzymatic activity observed for p.Ser633Trp variant of the α-L-iduronidase seems to lead to severe mucopolysaccharidosis type I phenotype, possibly associated with a perturbation of the structural dynamics in regions of the enzyme close to the active site.