IDUA Activity Research Articles

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.

Intra-Articular AAV9 α-l-Iduronidase Gene Replacement in the Canine Model of Mucopolysaccharidosis Type I

Mucopolysaccharidosis type I (MPS I), an inherited lysosomal storage disorder characterized by deficiency of α-l-iduronidase (IDUA) activity, causes multisystemic pathology due to sequelae of accumulated heparan and dermatan sulfates (HS and DS), the substrates of IDUA. Current treatments, though life-prolonging, inadequately address skeletal dysplasia and do not forestall progressive and painful degenerative joint disease. Previous studies demonstrated that intra-articular enzyme replacement cleared cellular lysosomal storage and reduced joint inflammation. Three nontolerized MPS I canines were studied to assess safety, efficacy, and durability of IDUA gene replacement therapy delivered via intra-articular injection. After baseline joint tissue biopsies, the right shoulder and stifle of each animal were injected in the intra-articular space with AAV9-IDUA and contralateral joints with AAV9-eGFP. Animals received either 5E11 or 5E12 vector genomes/joint. Necropsy was performed at 2- or 52-week postinjection. All animals tolerated injections without adverse effects. At two weeks, supraphysiologic IDUA enzyme activity was measured in AAV9-IDUA-treated but not AAV9-eGFP-treated synovium, with corresponding normalization of HS content and synoviocyte morphology. The AAV9-IDUA-treated cartilage had normal physiologic levels of IDUA enzyme, reduced but not normalized HS and DS levels compared to untreated MPS I cartilage, and healthy chondrocyte morphology. Liver IDUA transgene and IDUA enzyme activity were identified, as was serum IDUA activity which was 40% of wild-type serum enzyme activity. At 52-week postinjection, AAV9-IDUA-treated synovium and cartilage IDUA enzyme activity declined in both animals, corresponding to high tissue HS and DS levels and severe lysosomal storage. Liver and serum IDUA activity levels were undetectable. A dose-dependent serum anti-IDUA antibody response was observed which, together with loss of transgene with age, likely contributed to decline in tissue enzyme activity and treatment efficacy. Our study demonstrates successful proof-of-concept for intra-articular gene replacement therapy as a treatment for MPS-related joint dysplasia. Our observations suggest the possibility of multimodal gene replacement therapy to address multiple refractory manifestations of MPS I. Subsequent studies, in conjunction with immune tolerization and functional assessments of joint pathology, will investigate this possibility.

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.

Improving yield of a recombinant biologic in a Brassica hairy root manufacturing process.

Hairy root systems have proven to be a viable alternative for recombinant protein production. For recalcitrant proteins, maximizing the productivity of hairy root cultures is essential. The aim of this study was to optimize a Brassica rapa rapa hairy root process for secretion of alpha‐ l‐iduronidase (IDUA), a biologic of medical value. The process was first optimized with hairy roots expressing eGFP. For the biomass optimization, the highest biomass yields were achieved in modified Gamborg B5 culture medium. For the secretion induction, the optimized secretion media was obtained with additives (1.5 g/l PVP + 1 mg/l 2,4‐ d + 20.5 g/l KNO3) resulting in 3.4 fold eGFP secretion when compared to the non‐induced control. These optimized conditions were applied to the IDUA‐expressing hairy root clone, confirming that the highest yields of secreted IDUA occurred when using the defined additive combination. The functionality of the IDUA protein, secreted and intracellular, was confirmed with an enzymatic activity assay. A > 150‐fold increase of the IDUA activity was observed using an optimized secretion medium, compared with a non‐induced medium. We have proven that our B. rapa rapa hairy root system can be harnessed to secrete recalcitrant proteins, illustrating the high potential of hairy roots in plant molecular farming.

Establishment of Cutoff Values for Newborn Screening of Six Lysosomal Storage Disorders by Tandem Mass Spectrometry.

ObjectiveLysosomal storage disorders (LSDs) are becoming increasingly important in newborn screening, and tandem mass spectrometry (MS/MS) is widely used in newborn screening for LSDs through measurement of enzymatic activities in dried blood spots (DBSs). Overall, the determination of the cutoff value is important in such screening, and different laboratories have different methods of determining this value; most do not use a fixed cutoff value but rather calculate the corresponding batch cutoff value based on each batch of experimental data. In this study, we used MS/MS to screen for LSDs and sought to find an appropriate method to establish the cutoff value for LSD screening.MethodsA total of 38,945 samples from newborn blood tablets collected from various maternity hospitals in six cities in Shandong province, including Jinan, Dezhou, Heze, Linyi, Weifang, and Zibo, were tested using a Waters Xevo TQD tandem mass spectrometer; the experimental data were analyzed with MassLynx V4.1. The laboratory used 30% of the median GLA enzyme activity and 20% of the median ABG, ASM, GALC, IDUA, and GAA enzyme activities in every test as the cutoff values for that batch of experiments.ResultsThere were 254 suspicious positives in the initial screening test, including one case of Gaucher disease, one of Niemann-Pick disease, 47 of Krabbe disease, four of MPS-I, 21 of Fabry disease, and 180 of Pompe disease. After genetic screening, 11 children were diagnosed, including three with Pompe disease, three with Fabry disease, and five with Krabbe disease. In addition, the enzyme activity cutoff value of this experiment showed seasonal variation, which was initially believed to be related to the ambient temperature, such as the effect of ambient temperature on the human body or the temperature when the blood tablets dried naturally.ConclusionOverall, MS/MS can be used in LSD screening, and using different cutoff values in each batch of experiments is feasible. The ambient temperature might be a reason why the enzyme activity cutoff value has seasonal variation. More samples are needed to develop a method of determining cutoff values in laboratories.

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.

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

BackgroundAllogeneic 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.MethodsWe 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.ResultsWe 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.ConclusionsThe 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.)

GENE EDITING OF FIBROBLAST-LIKE SYNOVIOCYTES FROM MUCOPOLYSACCHARIDOSIS TYPE I MICE WITH THE CRISPR-CAS9 SYSTEM

Mucopolysaccharidosis type I (MPS I) is caused by alpha-L-iduronidase enzyme (IDUA) deficiency and lysosomal accumulation of glycosaminoglycans (GAGs). The accumulation of GAGs affects important cellular processes and triggers a cascade of responses altering cellular homeostasis and leading to multisystemic dysfunctions. In this disease, the bone/joint complications reduce mobility. Current treatments do not correct these complications. Therefore, it is necessary to look for new therapies. An alternative is gene editing using the CRISPRCas9 system. This work aims to evaluate the efficiency of cationic liposomes carrying plasmids with the CRISPRCas9 system plus a donor vector to replace IDUA in fibroblast-like synoviocytes. MPS I mouse fibroblast-like synoviocytes (FLS) were transfected with 1) Liposome/CRISPR+Donor IDUA or 2)Liposome/Donor IDUA or 3)Lipofectamine 3000® Lipofectamine/Donor IDUA or 4)Lipofectamine/CRISPR+Donor IDUA. The cells were maintained in culture and collected at 4 times (3, 7, 15, and 30 days) after transfection. IDUA activity and total proteins were measured at each of these times. The FLS from untreated MPS I and from normal mice were cultured in the same conditions and used as controls. The incubation of the complexes with FLS demonstrated high cell viability (over 70%) and no statistical difference (<i>p</i>>0.05) between treatments, and these results showed that the Liposome presented low cytotoxicity. At all times, the untreated FLS MPS I showed very low levels of IDUA. The treatment with Lipofectamine/CRISPR+Donor IDUA and Liposome/CRISPR+Donor IDUA always showed a significant increase in IDUA production (<i>p</i> < 0.0001 and <i>p</i><0.001). This increase was sustained for up to 30 days. The treatment with Lipofectamine/CRISPR+Donor IDUA reached an average of 4.0% (±0.26), of IDUA activity found in normal FLS, while the treatment with Liposome/CRISPR+Donor IDUA reached an average of 2.8% (±0.28). Both treatments were not significantly different from each other (<i>p</i>>0.05). Despite the enzyme levels not being normalized, due to cross-correction (enzyme exchange) between neighboring cells, an increase of 1% in the production of the enzyme could be enough to reduce the accumulation of glycosaminoglycans in lysosomes. This study demonstrates that the Liposome is appropriate for gene editing studies. New studies are being conducted in MPS I mice in vivo.

Longitudinal Analysis of Ocular Disease in Children with Mucopolysaccharidosis I after Hematopoietic Cell Transplantation.

Corneal clouding, causing visual impairment, is seen in nearly all patients with mucopolysaccharidosis type 1 (MPS-1). Hematopoietic cell transplantation (HCT) is able to stabilize disease in many organs. Residual disease in several tissues is being increasingly recognized, however. Data on the effect of HCT on ocular disease in patients with MPS-1 are contradictory. With this study, we aim to clarify the long-term effects of HCT on ocular disease in these patients. Best corrected visual acuity (BCVA), refraction, intraocular pressure (IOP), and slit-lamp biomicroscopic and fundoscopic examinations, including corneal clouding, were collected prospectively from 24 patients with MPS-1 who underwent HCT successfully between 2003 and 2018 (92% with >95% chimerism and normal enzyme activity after HCT). The course of corneal clouding and BCVA after HCT were analyzed using a linear mixed model. Other parameters studied were clinical phenotype, age at time of transplantation, and hematologic enzyme activity after transplantation. Outcomes of additional ophthalmologic tests were described. In addition, IDUA and α-galactosidase A (AGAL) enzyme activity and glycosaminoglycan (GAG) concentration in tear fluid were determined. Corneal clouding stabilized in the first years after HCT but increased rapidly beyond 3 years (P < .0001). BCVA and IOP also worsened over time (P=.01 and P < .0001, respectively). IDUA activity in tear fluid remained very low (P < .0001). After initial stabilization in the cornea, ongoing ocular disease and low IDUA activity in tear fluid is seen in patients with MPS-1 despite treatment with HCT, unveiling a weak spot of current standard therapy. New therapies that overcome these shortcomings are needed to improve the late outcomes of patients.

PS1221 LONG‐TERM EFFICACY AND SAFETY OF INOTERSEN FOR HEREDITARY TRANSTHYRETIN AMYLOIDOSIS: NEURO‐TTR OPEN‐LABEL EXTENSION 2‐YEAR UPDATE

Background:Hereditary transthyretin amyloidosis (hATTR) is a rare protein misfolding disorder that causes progressive and debilitating polyneuropathy. A randomized, controlled phase 3 trial (NEURO‐TTR; NCT01737398) demonstrated efficacy and safety of inotersen in patients with hATTR polyneuropathy (Benson 2018 NEJM).Aims:To provide an update on the long‐term efficacy and safety of inotersen, an antisense oligonucleotide inhibitor of transthyretin protein production, in patients with hereditary transthyretin amyloidosis (hATTR) with polyneuropathy.Methods:Patients who completed NEURO‐TTR were eligible to enroll in an ongoing open‐label extension (OLE) study (NCT02175004). We report an update on the long‐term efficacy and safety of inotersen in patients with hATTR polyneuropathy after 24 months in the OLE. Assessments included modified Neuropathy Impairment Score +7 neurophysiologic tests composite score (mNIS+7), Norfolk Quality of Life–Diabetic Neuropathy questionnaire total score (Norfolk QoL‐DN), Short Form 36 Health Survey version 2 (SF‐36v2) physical component summary (PCS), and safety monitoring.Results:Of 139 patients who completed NEURO‐TTR, 135 (97.1%) enrolled in the OLE. As of 5/31/2018, some patients were ongoing but had not yet completed 24 months in the OLE, and longest inotersen exposure in both studies was 5.2 years. Patients who switched from placebo to inotersen in the OLE demonstrated slowing of neurologic disease progression by mNIS+7 and Norfolk QoL‐DN as early as 6 months after starting inotersen (mean change from OLE baseline to month 6/year 2: 6.22/5.08 for mNIS+7 and 0.54/2.26 for Norfolk QoL‐DN). Patients who received inotersen for 39 months (15 months NEURO‐TTR + 24 months OLE) continued to show benefit (mean change from OLE baseline to year 2: 11.18 for mNIS+7 and 5.22 for Norfolk QoL‐DN). Patients who continued inotersen also showed stabilization of health‐related quality of life measured by SF‐36v2 PCS. No evidence of increased risk for grade 4 thrombocytopenia or severe renal events has been observed with increased exposure duration; no new safety concerns have been identified.Summary/Conclusion:In the OLE, inotersen improved, halted, or slowed progression of hATTR polyneuropathy, with greater stabilization observed in patients who initiated inotersen earlier.

PS1220 EX‐VIVO HEMATOPOIETIC STEM CELL GENE THERAPY (GT) FOR MUCOPOLYSACCHARIDOSIS TYPE I HURLER (MPSIH): PRELIMINARY RESULTS FROM A PHASE I/II CLINICAL STUDY

Background:MPSIH is a lysosomal storage disease caused by α‐L‐iduronidase (IDUA) deficiency that is associated with progressive multisystem morbidity including neurodevelopmental deterioration and severe orthopedic manifestations, leading to death in early childhood. Current therapeutic strategies include enzyme replacement therapy and allogeneic hematopoietic stem cell transplantation (HSCT), which however show limitations in preventing neurodevelopmental and orthopedic complications. Transplantation of genetically‐engineered autologous hematopoietic stem and progenitor cells (HSPC) is an attractive treatment option for MPSIH thanks to the possibility to obtain supraphysiologic levels of the missing enzyme in the hematopoietic progeny, thus allowing for increased cross‐correction.Aims:We recently opened a phase I/II clinical trial (NCT03488394) in children affected by MPSIH aimed at evaluating safety, tolerability and efficacy of the transplantation of autologous, IDUA lentiviral (LV)‐transduced CD34+ cells in MPSIH patients undergoing myeloablative conditioning.Methods:The study foresees the enrollment of 6 patients who lack a non‐heterozygous HLA‐matched donor and display an IQ/DQ&gt;70. Primary endpoints of safety include: overall survival, hematological engraftment and safety of drug product (DP) administration. Primary endpoint of efficacy is represented by IDUA activity in peripheral blood up to supraphysiologic levels at 1 year post‐gene therapy (GT). Treatment impact on nervous system, skeleton and other target organs is assessed by clinical and radiologic parameters, as well as by disease‐specific biomarkers.Results:By the submission deadline, two patients (24 and 14 months old) were treated and have a follow‐up of 7 and 1.5 months, respectively. Autologous HSPC were harvested after mobilization with G‐CSF and Plerixafor; the procedure was uneventful and resulted in cryopreserved DP of 24 and 14 million CD34+ cells/kg, respectively. Transduction efficiency in the drug products was above 80% using an optimized transduction protocol that preserved engraftment potential of ex vivo‐cultured HSPC in preclinical models. No adverse events related to DP infusion were recorded. Hematopoietic engraftment was fast in both patients; neutrophil engraftment occurred on day +17 and +22, respectively; platelet engraftment at day+15 with counts never falling below 30.000/ul in both patients. Importantly, both patients reached supraphysiologic levels of IDUA by day+14 that stabilized around 10 fold above the mean of the normal range, along with an in vivo vector copy number ranging from 2 to 4 in multiple hematopoietic lineages. At the 6 month follow‐up of patient 1, stable IDUA activity was detected in the cerebrospinal fluid, and urinary GAGs normalized. A third patient has been successfully mobilized resulting in a cryopreserved DP of 13 million CD34+ cells/kg and will be treated shortly. Other patients are currently under evaluation.Summary/Conclusion:Preliminary clinical results indicate that our optimized transduction protocol allows efficient HSPC transduction without compromising their engraftment potential. Treated patients showed rapid hematopoietic recovery, supraphysiologic IDUA levels in the peripheral blood and metabolic correction of the MPSI‐H enzyme defect in critical target tissues. Long term‐follow up and treatment of further patients are necessary to confirm that IDUA supraphysiologic levels result in improvement of neurological and orthopedic outcome as compared to allogeneic HSCT.

The North Carolina Experience with Mucopolysaccharidosis Type I Newborn Screening

To evaluate the performance of a 2-tiered newborn screening method for mucopolysaccharidosis type I (MPS I) in North Carolina. The screening algorithm included a flow injection analysis-tandem mass spectrometry assay as a first-tier screening method to measure α-L-iduronidase (IDUA) enzyme activity and Sanger sequencing of the IDUA gene on dried blood spots as a second-tier assay. The screening algorithm was revised to incorporate the Collaborative Laboratory Integrated Reports, an analytical interpretive tool, to reduce the false-positive rate. A medical history, physical examination, IDUA activity, and urinary glycosaminoglycan (GAG) analysis were obtained on all screen-positive infants. A total of 62 734 specimens were screened with 54 screen-positive samples using a cut-off of 15% of daily mean IDUA activity. The implementation of Collaborative Laboratory Integrated Reports reduced the number of specimens that screened positive to 19 infants. Of the infants identified as screen-positive, 1 had elevated urinary GAGs and a homozygous pathogenic variant associated with the severe form of MPS I. All other screen-positive infants had normal urinary GAG analysis; 13 newborns had pseudodeficiency alleles, 3 newborns had variants of unknown significance, and 2 had heterozygous pathogenic variants. An infant with severe MPS I was identified and referred for a hematopoietic stem cell transplant. Newborn IDUA enzyme deficiency is common in North Carolina, but most are due to pseudodeficiency alleles in infants with normal urinary GAG analysis and no evidence of disease. The pilot study confirmed the need for second-tier testing to reduce the follow-up burden.

Nasal Administration of Cationic Nanoemulsions as Nucleic Acids Delivery Systems Aiming at Mucopolysaccharidosis Type I Gene Therapy.

This study demonstrates the nasal administration (NA) of nanoemulsions complexed with the plasmid encoding for IDUA protein (pIDUA) as an attempt to reach the brain aiming at MPS I gene therapy. Formulations composed of DOPE, DOTAP, MCT (NE), and DSPE-PEG (NE-PEG) were prepared by high-pressure homogenization, and assessed in vitro on human fibroblasts from MPS I patients and in vivo on MPS I mice for IDUA production and gene expression. The physicochemical results showed that the presence of DSPE-PEG in the formulations led to smaller and more stable droplets even when submitted to dilution in simulated nasal medium (SNM). In vitro assays showed that pIDUA/NE-PEG complexes were internalized by cells, and led to a 5% significant increase in IDUA activity, besides promoting a two-fold increase in IDUA expression. The NA of pIDUA/NE-PEG complexes to MPS I mice demonstrated the ability to reach the brain, promoting increased IDUA activity and expression in this tissue, as well as in kidney and spleen tissues after treatment. An increase in serum IL-6 was observed after treatment, although with no signs of tissue inflammatory infiltrate according to histopathology and CD68 assessments. These findings demonstrated that pIDUA/NE-PEG complexes could efficiently increase IDUA activity in vitro and in vivo after NA, and represent a potential treatment for the neurological impairment present in MPS I patients.

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

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.

CRISPR-Cas9-mediated gene editing in human MPS I fibroblasts

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder (LSD). It is caused by mutations in the IDUA gene, which lead to the accumulation of the glycosaminoglycans dermatan and heparan sulfate. The CRISPR-Cas9 system is a new and powerful tool that allows gene editing at precise points of the genome, resulting in gene correction through the introduction and genomic integration of a wildtype sequence. In this study, we used the CRISPR-Cas9 genome editing technology to correct in vitro the most common mutation causing MPS I. Human fibroblasts homozygous for p.Trp402* (legacy name W402X) were transfected and analyzed for up to one month after treatment. IDUA activity was significantly increased, lysosomal mass was decreased, and next generation sequencing confirmed that a percentage of cells carried the wildtype sequence. As a proof of concept, this study demonstrates that CRISPR-Cas9 genome editing may be used to correct causative mutations in MPS I. List of abbreviationsUnlabelled TableCRISPRClustered Regularly Interspaced Short Palindromic RepeatsCasCRISPR-associatedERTenzyme replacement therapyHDRhomology-directed repairHSCThematopoietic stem cell transplantationIDUAalpha-L-iduronidaseLSDlysosomal storage disorderMPS IMucopolysaccharidosis type I

Intra-articular nonviral gene therapy in mucopolysaccharidosis I mice

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.

Gene editing of MPS I human fibroblasts by co-delivery of a CRISPR/Cas9 plasmid and a donor oligonucleotide using nanoemulsions as nonviral carriers

Mucopolysaccharidosis type I (MPS I) is an inherited disease caused by the deficiency of alpha-L-iduronidase (IDUA). This study shows the use of nanoemulsions co-complexed with the plasmid of CRISPR/Cas9 system and a donor oligonucleotide aiming at MPS I gene editing in vitro. Nanoemulsions composed of MCT, DOPE, DOTAP, DSPE-PEG, and water were prepared by high-pressure homogenization. The DNA was complexed by adsorption (NA) or encapsulation (NE) of preformed DNA/DOTAP complexes with nanoemulsions at +4/−1 charge ratio. The incubation in pure DMEM or supplemented with serum showed that the complexation with DNA was stable after 1 h of incubation, but the complexes tended to release the adsorbed DNA after 24 h of incubation, while the encapsulated DNA remained complexed in the oil core of the nanoemulsions even 48 h after incubation with DMEM. The treatment of MPS I patient’s fibroblasts homozygous for the p.Trp402∗ mutation led to a significant increase in IDUA activity at 2, 15, and 30 days when compared to MPS I untreated fibroblasts. Flow cytometry and confocal microscopy demonstrated that there was a reduction in the area of lysosomes to values similar to normal, an indicator of correction of the cellular phenotype. These results show that the nanoemulsions co-complexed with the CRISPR/Cas9 system and a donor oligonucleotide could effectively transfect MPS I p.Trp402∗ patient’s fibroblasts, as well as enable the production of IDUA, and represent a potential new treatment option for MPS I.

Optimization of alginate microcapsules containing cells overexpressing α-l-iduronidase using Box-Behnken design

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive disease caused by deficiency of α-l-iduronidase (IDUA), which results in the lysosomal accumulation of glycosaminoglycans (GAG) leading to widespread clinical manifestations. The microencapsulation of IDUA overexpressing recombinant cells has been considered as a promising strategy for the treatment of MPS I. This study aimed at the optimization of alginate microcapsules containing recombinant BHK (Baby Hamster Kidney) cells (rBHK) overexpressing IDUA produced by electrostatic extrusion technique. The alginate microcapsule (MC-A) optimization study was carried out by means of an experimental Box-Behnken Design that allowed the simultaneous evaluation of the influence of voltage (kV), alginate/cell suspension flow (mL/h), and alginate concentration (%) on size and IDUA activity. The optimal conditions of voltage (10kV), flow (25mL/h), and alginate concentration (1.3%) made possible to obtain the smallest microcapsules showing the highest IDUA activity. After optimization, the microcapsules were sequentially coated with PLL and alginate (MC-APA) to increase their stability. MC-A and MC-APA presented monodisperse populations (span<1.22) with an average diameter of less than 350μm. The coating increased the mechanical stability of MC-APA by about 6-fold and modulated the permeability to the enzyme. Surface analyzes of MC-APA showed the presence of PLL bands, suggesting that the last alginate layer appears to have only partially coated the PLL. After 30days of subcutaneous implantation of the MC-APA microcapsules containing rBHK cells in a MPS I murine model, a significant increase in IDUA activity was observed in the skin near the implant. Histological analysis revealed an inflammatory infiltrate at the application site, which did not prevent the release of the enzyme under the conditions evaluated. Taken together, the overall results demonstrate the feasibility of MC-APA as a potential alternative for local treatment of MPS I.

Alpha-l-iduronidase and arylsulfatase B in dried blood spots on filter paper: Biochemical parameters and time stability

BackgroundThe goal of this study was to assess the biochemical parameters of the enzymes α-l-iduronidase (IDUA) and arylsulfatase B (ASB), which are deficient in mucopolysaccharidosis (MPS) I and VI, respectively, in dried blood spot (DBS) samples impregnated on filter paper. Methods and resultsThe optimal pH, Km, and Vmax of IDUA and ASB in DBS are hereby presented. After these analyses, the reference values for the activities of these enzymes in DBS with cutoff of 3.65nmol/h/mL for IDUA and 6.80nmol/h/mL for ASB were established. The research also showed that the stability (21days) of the IDUA activity is lower than ASB, which maintained its enzymatic activity stable up until 60days of analysis, after impregnating the filter paper with blood. ConclusionCurrently, DBS ensures important advantages in handling storage and transportation of samples with respect to neonatal screening programs. This study contributes to characterizing and differentiating the biochemistry of deficient enzymes in MPSs I and VI of DBS samples.

93. Development of AAVidua Vector for the Treatment of Cornea Associated Vision Loss in Bone Marrow Treated-Hurler Patients

Mucopolysaccharidosis type 1 (MPS1), also known as Hurler syndrome, is a genetic lysosomal storage disease that results from the loss-of-function mutations present on the L-iduronidase (IDUA) gene. As a consequence, glycosaminoglycans accumulate aberrantly in lysosomes in multiple organs, leading to hepatosplenomegaly, dwarfism, mental and psychomotor retardation, life-threatening cardiac and pulmonary complications, several skeletal and ocular manifestations and nervous system problems. Untreated pediatric patients often die at 5-10 years of age from progressive heart and lung involvement. Since the early 1980s, successful treatment of MPS1 patients with allogeneic hematopoietic stem cell transplantation after myeloablative chemotherapy has been established. The benefits of this therapy rely on cross-correction of IDUA deficient cells with functional IDUA protein produced by a donor cell circulating through the blood and also from engraftment of donor-derived glial cells in the MPS1 patient brain. As a result, if the stem cell transplantation is performed before two years old, the individual manifest cardiac, liver, pulmonary and neurological improvement as well as his lifetime is significantly prolonged. However, still >30% of the patients have progressive corneal clouding, which leads to blindness. Cornea transplantation is not an option for patients with severe hurler disease and the ones that receive a cornea transplant are at the risk of developing future cloudiness. Recently, we have developed an adeno-associated virus (AAV) capable of delivering idua cDNA to MPS1 patient fibroblasts and restoring IDUA protein function. Furthermore, immunohistochemistry and functional analysis of intrastromal IDUA protein in a normal human cornea showed low levels of the protein. Even though the levels of IDUA that are required to correct the loss of sight in MPS1 patients are relatively low, administration of a >50-fold increase in IDUA activity (over wild type levels) with gene therapy does not result in any detectable cellular toxicity. We then investigated AAV serotype tropism by incubating human cornea with different AAV viral capsids carrying AAV-CMV-eGFP. From this experiment, we have identified certain capsids with higher levels of transduction and GFP expression on the human cornea. Moreover, we determined the optimal injection volume required for complete coverage of the corneal center area, which is sufficient for allowing regain of the vision. Considering that: 1) the eye is an easily accessible, immune-privileged organ; 2) intrastromal injections are commonly performed at the clinic to treat fungal keratitis; 3) gene therapy for eye diseases has been performed for nearly two decades, and 4) there are 127 registered clinical trials currently taken place for gene therapy in the eye; we foresee that injection of AAV delivering idua cDNA directly to the cornea will likely reverse the MPSl-associated vision loss and provide a better quality of life for the patient.