Gabriela Pasqualim

Emerging drugs for the treatment of mucopolysaccharidoses.

ABSTRACT Introduction: Despite being reported for the first time almost one century ago, only in the last few decades effective have treatments become available for the mucopolysaccharidoses (MPSs), a group of 11 inherited metabolic diseases that affect lysosomal function. These diseases are progressive, usually severe, and, in a significant number of cases, involve cognitive impairment. Areas covered: This review will not cover established treatments such as bone marrow/hematopoietic stem cell transplantation and classic intravenous enzyme replacement therapy (ERT), whose long-term outcomes have already been published (MPS I, MPS II, and MPS VI), but it instead focuses on emerging therapies for MPSs. That includes intravenous ERT for MPS IVA and VII, intrathecal ERT, ERT with fusion proteins, substrate reduction therapy, gene therapy, and other novel approaches. Expert opinion: The available treatments have resulted in improvements for several disease manifestations, but they still do not represent a cure for these diseases; thus, it is important to develop alternative methods to approach the unmet needs (i.e. bone disease, heart valve disease, corneal opacity, and central nervous system (CNS) involvement). The work in progress with novel approaches makes us confident that in 2017, when MPS will commemorate 100 years of its first report, we will be much closer to an effective cure for these challenging conditions.

Fabry disease: a new approach for the screening of females in high-risk groups.

OBJECTIVE Fabry disease (FD) is a rare X-linked inborn error of metabolism caused by deficient activity of lysosomal α-galactosidase A (α-GAL). Due to random X inactivation, α-GAL activity in heterozygous females ranges from very low to overlapping normal values. Determining this specific range and altering assays cutoffs could become a valuable tool for minimizing the need in DNA sequencing for screening of all potential carriers. Therefore, the aim of this study was to establish the range of enzyme in dried blood spots (DBS), plasma and leukocytes that suggests carrier status for FD. DESIGN AND METHODS α-GAL gene was sequenced in 453 women with clinical suspicion and/or positive family history of FD. This data was compared to the α-GAL activity measured in DBS (dried blood spots) and/or plasma and/or leukocytes. RESULTS About 12% of the samples had pathogenic mutations (c.30_32delG, c.718_719delAA, p.R118C, p.S126G, p.Y152X, p.A156D, p.C202Y, p.N215S, p.P259R, p.D264Y, p.V269M, p.R342Q and p.R356W). When compared to genotype, DBS was the least reliable biochemical test for screening, with very low specificity. Plasma and leukocyte activities presented high AUC in ROC curve analysis, both over 84%. When cutoffs were altered to identify all carriers, leukocyte specificity was higher than that of plasma (35.2% and 27.6%, respectively). Moderated correlation and agreement coefficients were found between them, which reinforces the need for using both data combined. CONCLUSION A combined approach involving plasma and leukocyte α-GAL activities, with distinct cutoffs for men and women, could represent a more accurate, faster and less expensive tool to screen women for FD in high-risk groups in middle- and low-income countries.

Current molecular genetics strategies for the diagnosis of lysosomal storage disorders

Lysosomal storage disorders (LSDs) are a group of almost 50 monogenic diseases characterized by mutations causing deficiency of lysosomal enzymes or non-enzyme proteins involved in transport across the lysosomal membrane, protein maturation or lysosomal biogenesis. Usually, affected patients are normal at birth and have a progressive and severe disease with high morbidity and reduced life expectancy. The overall incidence of LSDs is usually estimated as 1:5000, but newborn screening studies are indicating that it could be much higher. Specific therapies were already developed for selected LSDs, making the timely and correct diagnosis very important for successful treatment and also for genetic counseling. In most LSD cases the biochemical techniques provide a reliable diagnosis. However, the identification of pathogenic mutations by genetic analysis is being increasingly recommended to provide additional information. In this paper we discuss the conventional methods for genetic analysis used in the LSDs [restriction fragment length polymorphism (RFLP), amplification-refractory mutation system (ARMS), single strand conformation polymorphism (SSCP), denaturing high performance liquid chromatography (dHPLC), real-time polymerase chain reaction, high resolution melting (HRM), multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing] and also the newer approaches [massive parallel sequencing, array comparative genomic hybridization (CGH)].

Effects of enzyme replacement therapy started late in a murine model of mucopolysaccharidosis type I

Mucopolysaccharidosis type I (MPS I) is a progressive disorder caused by deficiency of α-L-iduronidase (IDUA), which leads to storage of heparan and dermatan sulphate. It is suggested that early enzyme replacement therapy (ERT) leads to better outcomes, although many patients are diagnosed late and don’t receive immediate treatment. This study aims to evaluate the effects of late onset ERT in a MPS I murine model. MPS I mice received treatment from 6 to 8 months of age (ERT 6–8mo) with 1.2mg laronidase/kg every 2 weeks and were compared to 8 months-old wild-type (Normal) and untreated animals (MPS I). ERT was effective in reducing urinary and visceral GAG to normal levels. Heart GAG levels and left ventricular (LV) shortening fraction were normalized but cardiac function was not completely improved. While no significant improvements were found on aortic wall width, treatment was able to significantly reduce heart valves thickening. High variability was found in behavior tests, with treated animals presenting intermediate results between normal and affected mice, without correlation with cerebral cortex GAG levels. Cathepsin D activity in cerebral cortex also did not correlate with behavior heterogeneity. All treated animals developed anti-laronidase antibodies but no correlation was found with any parameters analyzed. However, intermediary results from locomotion parameters analyzed are in accordance with intermediary levels of heart function, cathepsin D, activated glia and reduction of TNF-α expression in the cerebral cortex. In conclusion, even if started late, ERT can have beneficial effects on many aspects of the disease and should be considered whenever possible.

Investigation of newborns with abnormal results in a newborn screening program for four lysosomal storage diseases in Brazil

Lysosomal storage diseases (LSDs) are genetic disorders, clinically heterogeneous, mainly caused by defects in genes encoding lysosomal enzymes that degrade macromolecules. Several LSDs already have specific therapies that may improve clinical outcomes, especially if introduced early in life. With this aim, screening methods have been established and newborn screening (NBS) for some LSDs has been developed. Such programs should include additional procedures for the confirmation (or not) of the cases that had an abnormal result in the initial screening. We present here the methods and results of the additional investigation performed in four babies with positive initial screening results in a program of NBS for LSDs performed by a private laboratory in over 10,000 newborns in Brazil. The suspicion in these cases was of Mucopolysaccharidosis I - MPS I (in two babies), Pompe disease and Gaucher disease (one baby each). One case of pseudodeficiency for MPS I, 1 carrier for MPS I, 1 case of pseudodeficiency for Pompe disease and 1 carrier for Gaucher disease were identified. This report illustrates the challenges that may be encountered by NBS programs for LSDs, and the need of a comprehensive protocol for the rapid and precise investigation of the babies who have an abnormal screening result.

Identification of mutations in Colombian patients affected with Fabry disease

Fabry Disease (FD) is an X-linked inborn error of glycosphingolipid catabolism, caused by a deficiency of the lisosomal α-galactosidase A (AGAL). The disorder leads to a vascular disease secondary to the involvement of kidney, heart and the central nervous system. The mutation analysis is a valuable tool for diagnosis and genetic counseling. Although more than 600 mutations have been identified, most mutations are private. Our objective was to describe the analysis of nine Colombian patients with Fabry disease by automated sequencing of the seven exons of the GLA gene. Two novel mutations were identified in two patients affected with the classical subtype of FD, in addition to other 6 mutations previously reported. The present study confirms the heterogeneity of mutations in Fabry disease and the importance of molecular analysis for genetic counseling, female heterozygotes detection as well as therapeutic decisions.

p.L18P: a novel IDUA mutation that causes a distinct attenuated phenotype in mucopolysaccharidosis type I patients

Mucopolysaccharidosis type I is a rare autosomal recessive disorder caused by deficiency of α‐l‐iduronidase (IDUA) which leads to a wide spectrum of clinical severity. Here, we describe the case of four male patients who present the previously undescribed p.L18P mutation. Patient 1 (p.L18P/p.L18P) presents, despite multiple joint contractures, an attenuated phenotype. Patient 2 (p.L18P/p.W402X) was diagnosed at 4 years of age with bone dysplasia, coarse facies, limited mobility, claw hands and underwent bilateral carpal tunnel surgery at 6 years of age. Patients 3 and 4 (both p.L18P/p.L18P) are brothers. Patient 3 was diagnosed at 4 years of age, when presented claw hands, lower limb and shoulder pain, restricted articular movement and bilateral carpal tunnel syndrome. Patient 4 was diagnosed at 17 months of age when presented lower limb pain at night, respiratory allergy and repeated upper airways infections. Bioinformatics analysis indicates that p.L18P mutation reduces the signal peptide to 25 amino acids and alters its secondary structure. In conclusion, we report a new IDUA variant that alters the structure of the signal peptide, which likely impairs transport to lysosomes. Moreover, it leads to a distinct attenuated phenotype with mainly bone and cartilage symptoms, without visceromegalies, heart disease, or cognitive impairment.

Analysis of cDNA molecules is not suitable for the molecular diagnosis of Mucopolysaccharidosis type I.

Nonsense-mediated decay (NMD) is a mechanism of the recognition and degradation of messenger RNA containing a premature stop codon. Nonsense mutations are the main mutations that lead to Mucopolysaccharidosis type I. To determine the effect of NMD on correct genotyping based on cDNA sequencing, we standardized the sequencing from alpha-L-iduronidase gene cDNA molecules and validated this process for a group of patients whose mutations had been previously identified by DNA analysis. Although the whole gene could be amplified in 5 polymerase chain reactions, cDNA proved unsuitable for molecular analysis as patients bearing splice site and nonsense mutations were not genotyped.

In vivo genome editing of mucopolysaccharidosis I mice using the CRISPR/Cas9 system

ABSTRACT Mucopolysaccharidosis type I (MPS I) is a multisystemic disorder caused by the deficiency of alpha‐L‐iduronidase (IDUA) that leads to intracellular accumulation of glycosaminoglycans (GAG). In the present study we aimed to use cationic liposomes carrying the CRISPR/Cas9 plasmid and a donor vector for in vitro and in vivo MPS I gene editing, and compare to treatment with naked plasmids. The liposomal formulation was prepared by microfluidization. Complexes were obtained by the addition of DNA at +4/−1 charge ratio. The overall results showed complexes of about 110 nm, with positive zeta potential of +30 mV. The incubation of the complexes with fibroblasts from MPS I patients led to a significant increase in IDUA activity and reduction of lysosomal abnormalities. Hydrodynamic injection of the liposomal complex in newborn MPS I mice led to a significant increase in serum IDUA levels for up to six months. The biodistribution of complexes after hydrodynamic injection was markedly detected in the lungs and heart, corroborating the results of increased IDUA activity and decreased GAG storage especially in these tissues, while the group that received the naked plasmids presented increased enzyme activity especially in the liver. Furthermore, animals treated with the liposomal formulation presented improvement in cardiovascular parameters, one of the main causes of death observed in MPS I patients. We conclude that the IDUA production in multiple organs had a significant beneficial effect on the characteristics of MPS I disease, which may bring hope to gene therapy of Hurler patients. Graphical abstract Figure. No Caption available. HighlightsLiposomes are efficient carriers for CRISPR/Cas9 system.Liposomal CRISPR/Cas9 complexes showed efficient gene editing of MPS I fibroblasts.Treatment of newborn MPS I mice promoted high serum IDUA levels for up to 6 months.Treatment with liposomal complexes was more efficient than Naked CRISPR/Cas9.Liposomal CRISPR/Cas9‐treated mice improved GAG accumulation and cardiac function.

Intra-articular nonviral gene therapy in mucopolysaccharidosis I mice

ABSTRACT Mucopolysaccharidosis type I (MPS I) is caused by the lysosomal accumulation of glycosaminoglycans (GAGs) due to the deficiency of the enzyme alpha‐L‐iduronidase (IDUA). Currently available treatments may improve several clinical manifestations, but they have limited effects on joint disease, resulting in persistent orthopedic complications and impaired mobility. Thus, this study aimed to perform an intra‐articular administration of cationic nanoemulsions complexed with the plasmid encoding for the IDUA protein (pIDUA) targeting MPS I gene therapy for the synovial joints. Formulations composed of DOPE, DOTAP, MCT (NE), and DSPE‐PEG (NE‐PEG) were prepared by high‐pressure homogenization, and the pIDUA plasmid was associated by adsorption onto the surface of nanoemulsions (pIDUA/NE or pIDUA/NE‐PEG). The physicochemical characterization showed that the presence of DSPE‐PEG in pIDUA/NE‐PEG formulations led to small and highly stable droplets even when incubated with simulated synovial fluid (SSF), when compared to the non‐pegylated complexes (pIDUA/NE). Uptake by fibroblast‐like synoviocytes (FLS) was demonstrated, and high cell viability (70%) in addition with increased IDUA activity (2.5% of normal) were observed after incubation with pIDUA/NE‐PEG. The intra‐articular injection of pIDUA/NE‐PEG complexes in MPS I mice showed that the complexes were localized in the joints, were able to transfect synovial cells, and thus promoted an increase in IDUA activity and expression in the synovial fluid, with no significant activity in other tissues (kidney, liver, lung, and spleen). The overall results demonstrated a contained, safe, tolerable, and effective in situ approach of nonviral intra‐articular gene therapy targeting the reduction or prevention of the debilitating orthopedic complications of MPS I disorder.