Mucopolysaccharidosis type VII (Sly syndrome) - What do we know?

Mucopolysaccharidosis type VII (MPS VII) is an ultra-rare, life-threatening, progressive disease caused by genetic mutations that affect lysosomal storage/function. MPS VII has an estimated prevalence of <1:1,000,000 and accounts for <3% of all MPS diagnoses. Given the rarity of MPS VII, comprehensive information on the disease is limited and we present a review of the current understanding. In MPS VII, intracellular glycosaminoglycans accumulate due to a deficiency in the lysosomal enzyme that is responsible for their degradation, ß-glucuronidase, which is encoded by the GUSB gene. MPS VII has a heterogeneous presentation. Features can manifest across multiple systems and can vary in severity, age of onset and progression. The single most distinguishing clinical feature of MPS VII is non-immune hydrops fetalis (NIHF), which presents during pregnancy. MPS VII usually presents within one month of life and become more prominent at 3 to 4 years of age; key features are skeletal deformities, hepatosplenomegaly, coarse facies, and cognitive impairment, although phenotypic variation is a hallmark. Current treatments include hematopoietic stem cell transplantation and enzyme replacement therapy with vestronidase alfa. Care should be individualized for each patient. Development of consensus guidelines for MPS VII management and treatment is needed, as consolidation of expert knowledge and experience (for example, through the MPS VII Disease Monitoring Program) may provide a significant positive impact to patients.

Self-complementary AAV vector therapy for treating corneal cloudiness of mucopolysaccharidosis type VII (MPS VII).

PURPOSE: To design a novel efficacious scAAV-Gusb viral vector for treating Mucopolysaccharidosis Type VII (MPS VII) caused by a mutation in the ß-Glu gene (Gusb allele). METHODS: ß-Glu expression of single-stranded AAV-Gusb (ssAAV-Gusb) and self-complementary AAV (scAAV-Gusb) vectors are tested with cultured murine Gusb fibroblasts. The scAAV-Gusb vector was chosen in further studies to prolong the life span and treat corneal pathology of Gusb mice via intrahepatic injection of neonates and intrastromal injection in adults, respectively. Corneal pathology was studied using HRT2 in vivo confocal microscope and histochemistry in mice corneas. RESULTS: Both ssAAV-Gusb and scAAV-Gusb vectors expressed murine ß-Glu in cultured Gusb fibroblasts. The scAAV-Gusb vector had higher transduction efficiency than the ssAAV-Gusb vector. To prolong the life span of Gusb mice, neonates (3 days old) were administered with scAAV-Gusb virus via intrahepatic injection. The treatment improves the survival rate of Gusb mice, prolonging the median survival rate from 22.5 weeks (untreated) to 50 weeks (treated). Thereafter, we determined the efficacy of the scAAV-Gusb virus in ameliorating corneal cloudiness observed in aged Gusb mice. Both corneal cloudiness and stroma thickness decreased, and there was the presence of ß-Glu enzyme activity in the Gusb corneas receiving scAAV-Gusb virus associated with morphology change of amoeboid stromal cells in untreated to characteristic dendritic keratocytes morphology after 4-12 weeks of scAAV-Gusb virus injection. CONCLUSION: Intrahepatic injection of scAAV-Gusb is efficacious in prolonging the life span of Gusb mice, and intrastromal injection can ameliorate corneal phenotypes. Both strategies can be adapted for treating other MPS.

Prenatal diagnosis of mucopolysaccharidosis type VII facilitating treatment in neonatal period.

Duo exome testing was performed on a fetus conceived via in vitro fertilization with an egg donor. The fetus presented with non-immune hydrops fetalis (NIHF) at 20 + 0 weeks gestation. Two variants were detected in the GUSB gene. Biallelic pathogenic variants cause mucopolysaccharidosis type VII (MPS-VII), which can present with NIHF prenatally. At the time of analysis and initial report, one variant was classified as likely pathogenic and the other as of uncertain clinical significance. Biochemical testing of the amniotic fluid supernatant showed elevated glycosaminoglycans and low ß-glucuronidase activity consistent with the diagnosis of MPS-VII. This evidence allowed the upgrade of the pathogenicity for both variants, confirming the diagnosis of MPS-VII. The infant was born at 36 + 5 weeks and enzyme replacement therapy (ERT) using vestronidase was initiated at 20 days with planning for hematopoietic stem cell transplant ongoing. The ERT therapy has been well tolerated, with decreasing quantitative urine glycosaminoglycans. Long-term follow up is required to determine whether treatment has been successful. This case demonstrates the utility of alternative testing methods to clarify the pathogenicity of variants and the clinical utility of obtaining a diagnosis antenatally in facilitating treatment in the neonatal period, and specifically highlights MPS-VII as a treatable cause of NIHF.

Long-term efficacy and safety of vestronidase alfa enzyme replacement therapy in pediatric subjects < 5 years with mucopolysaccharidosis VII.

Mucopolysaccharidosis (MPS) VII is an ultra-rare, autosomal-recessive, metabolic disease caused by a deficiency of ß-glucuronidase, a lysosomal enzyme that hydrolyzes glycosaminoglycans (GAGs), including dermatan sulfate (DS), chondroitin sulfate, and heparan sulfate (HS). ß-glucuronidase deficiency leads to progressive accumulation of undegraded GAGs in lysosomes of affected tissues, which may cause hydrops fetalis, short stature, hepatosplenomegaly, and cognitive impairment. An open-label, multicenter, phase II study was conducted in 8 pediatric subjects <5 years of age with MPS VII. Subjects received the recombinant human ß-glucuronidase vestronidase alfa 4 mg/kg by intravenous infusion every other week for 48 weeks (treatment period). Those who completed the 48-week treatment were offered to continue treatment with vestronidase alfa 4 mg/kg for up to 240 weeks or until withdrawal of consent, discontinuation, or study termination (continuation period). The level of GAG excreted in urine (uGAG) above normal has been shown to correlate with disease severity and clinical outcomes in MPS diseases. Therefore, the primary efficacy endpoint of this study was to determine the mean percentage change in uGAG DS excretion from baseline to week 48. Statistically significant reductions in uGAG DS from baseline were observed at each visit (p < 0.0001), with a least square mean (standard error) percentage change of -60% (6.6) at week 4 (first post-baseline assessment) and -61% (6.41) at week 48 (final assessment during treatment period). Secondary efficacy endpoints included change from baseline to week 48 in growth and hepatosplenomegaly. Positive trends were observed toward increased standing height Z-score (mean [standard deviation] at baseline, -2.630 [1.17], n = 8; at week 48, -2.045 [0.27], n = 7) and growth velocity (mean [SD] Z-score at baseline, -2.59 [1.49], n = 4; at week 48, -0.39 [2.10], n = 4; p = 0.27). Hepatomegaly was resolved in 3 of 3 subjects assessed by ultrasound and in 5 of 6 subjects assessed by physical examination; splenomegaly was resolved in 1 of 3 subjects assessed by ultrasound and in 2 of 2 subjects assessed by physical examination. There were no new safety signals identified during this study. Mild-to-moderate infusion-associated reactions occurred in 4 (50%) subjects. In conclusion, long-term vestronidase alfa treatment demonstrated a rapid and sustained reduction in uGAGs, maintained growth, and improved hepatosplenomegaly in pediatric subjects with MPS VII <5 years of age. Trial registration: NCT02418455.

Adipose deficiency and aberrant autophagy in a Drosophila model of MPS VII is corrected by pharmacological stimulators of mTOR.

Mucopolysaccharidosis type VII (MPS VII) is a recessively inherited lysosomal storage disorder caused due to ß-glucuronidase (ß-GUS) enzyme deficiency. Prominent clinical symptoms include hydrops fetalis, musculoskeletal deformities, neurodegeneration and hepatosplenomegaly leading to premature death in most cases. Apart from these, MPS VII is also characterized as adipose storage deficiency disorder although the underlying mechanism of this lean phenotype in the patients or ß-GUS-deficient mice still remains a mystery. We addressed this issue using our recently developed Drosophila model of MPS VII (the CG2135-/- fly), which also exhibited a significant loss of body fat. We report here that the lean phenotype of the CG2135-/- larvae is due to fewer number of adipocytes, smaller lipid droplets and reduced adipogenesis. Our data further revealed that there is an abnormal accumulation of autophagosomes in the CG2135-/- larvae due to autophagosome-lysosome fusion defect. Decreased lysosome-mediated turnover also led to attenuated mTOR activity in the CG2135-/- larvae. Interestingly, treatment of the CG2135-/- larvae with mTOR stimulators, 3BDO or glucose, led to the restoration of mTOR activity with simultaneous correction of the autophagy defect and adipose storage deficiency. Our finding thus established a hitherto unknown mechanistic link between autophagy dysfunction, mTOR downregulation and reduced adiposity in MPS VII.

The first mucopolysaccharidosis type VII in a Taiwanese girl: A case report and review of the literature.

The present study included the first case of mucopolysaccharidosis (MPS) type VII in Taiwan. During pregnancy, the patient was diagnosed with hydrops fetalis and had ascites aspiration 4 times. In the following years, she presented gradually with chronic lung disease, developmental delay, short stature, dysmorphic features of coarse face, macroglossia and pigeon chest with scoliosis. Upon referral at age 4 years, she had corneal clouding, mild limitation of range of motion (ROM) and hepatosplenomegaly. X-ray showed paddle ribs and dysplastic vertebral bodies. MPS was suspected and urine glycosaminoglycans (GAGs) elevated were noted. The leukocyte enzymatic analyses for MPS I, MPS II, MPS IIIB, MPS IVA, and MPS VI were all normal. Afterward, the molecular analysis showed two heterozygous genetic variants of c.104C > A and c.1454C > T in trans in the GUSB gene (NM_000181.4) which were the causes for MPS VII. Then, we checked the leukocyte ß-glucuronidase activity for MPS VII and showed extremely low, therefore confirmed the diagnosis. Clinicians should increase the awareness on the early signs of MPS to have a prompt diagnosis and offer the correct treatment like enzyme replacement therapy (ERT) as early as possible.

Vestronidase alfa: Recombinant human ß-glucuronidase as an enzyme replacement therapy for MPS VII.

Mucopolysaccharidosis VII (MPS VII) is a rare lysosomal storage disease characterized by a deficiency in the enzyme ß-glucuronidase that has previously been successfully treated in a mouse model with enzyme replacement therapy. Here, we present the generation of a novel, highly sialylated version of recombinant human ß-glucuronidase (rhGUS), vestronidase alfa, that has high uptake, resulting in an improved enzyme replacement therapy for the treatment of patients with MPS VII. In vitro, vestronidase alfa has 10-fold more sialic acid per mole of rhGUS monomer than a prior rhGUS version (referred to as GUS 43/44) and demonstrated very high affinity at ~1 nM half maximal uptake in human MPS VII fibroblasts. Vestronidase alfa has a longer enzymatic half-life after uptake into fibroblasts compared with other enzymes used as replacement therapy for MPS (40 days vs 3 to 4 days, respectively). In pharmacokinetic and tissue distribution experiments in Sprague-Dawley rats, intravenous administration of vestronidase alfa resulted in higher serum rhGUS levels and enhanced ß-glucuronidase activity distributed to target tissues. Weekly intravenous injections of vestronidase alfa (0.1 mg/kg to 20 mg/kg) in a murine model of MPS VII demonstrated efficient enzyme delivery to all tissues, including bone and brain, as well as reduced lysosomal storage of glycosaminoglycans (GAGs) in a dose-dependent manner, resulting in increased survival after 8 weeks of treatment. Vestronidase alfa was well-tolerated and demonstrated no toxicity at concentrations that reached 5-times the proposed clinical dose. In a first-in-human phase 1/2 clinical trial, a dose-dependent reduction in urine GAG levels was sustained over 38 weeks of treatment with vestronidase alfa. Together, these results support the therapeutic potential of vestronidase alfa as an enzyme replacement therapy for patients with MPS VII.

Tolerance induction and microglial engraftment after fetal therapy without conditioning in mice with Mucopolysaccharidosis type VII.

Mucopolysaccharidosis type VII (MPS7) is a lysosomal storage disorder (LSD) resulting from mutations in the ß-glucuronidase gene, leading to multiorgan dysfunction and fetal demise. While postnatal enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation have resulted in some phenotypic improvements, prenatal treatment might take advantage of a unique developmental window to penetrate the blood-brain barrier or induce tolerance to the missing protein, addressing two important shortcomings of postnatal therapy for multiple LSDs. We performed in utero ERT (IUERT) at E14.5 in MPS7 mice and improved survival of affected mice to birth. IUERT penetrated brain microglia, whereas postnatal administration did not, and neurological testing (after IUERT plus postnatal administration) showed decreased microglial inflammation and improved grip strength in treated mice. IUERT prevented antienzyme antibody development even after multiple repeated postnatal challenges. To test a more durable treatment strategy, we performed in utero hematopoietic stem cell transplantation (IUHCT) using congenic CX3C chemokine receptor 1-green fluorescent protein (CX3CR1-GFP) mice as donors, such that donor-derived microglia are identified by GFP expression. In wild-type recipients, hematopoietic chimerism resulted in microglial engraftment throughout the brain without irradiation or conditioning; the transcriptomes of donor and host microglia were similar. IUHCT in MPS7 mice enabled cross-correction of liver Kupffer cells and improved phenotype in multiple tissues. Engrafted microglia were seen in chimeric mice, with decreased inflammation near donor microglia. These results suggest that fetal therapy with IUERT and/or IUHCT could overcome the shortcomings of current treatment strategies to improve phenotype in MPS7 and other LSDs.

C-Type Natriuretic Peptide Restores Growth Impairment Under Enzyme Replacement in Mice With Mucopolysaccharidosis VII.

Growth impairment in mucopolysaccharidoses (MPSs) is an unresolved issue as it is resistant to enzyme replacement therapy (ERT) and growth hormone therapy. C-type natriuretic peptide (CNP) is a promising agent that has growth-promoting effects. Here we investigate the effects of CNP on growth impairment of MPSs using Gusbmps-2J mice, a model for MPS type VII, with combination therapy of CNP and ERT by hydrodynamic gene delivery. Although monotherapies were not sufficient to restore short statures of treated mice, combination therapy resulted in successful restoration. The synergistic effects of CNP and ERT were not only observed in skeletal growth but also in growth plates. ERT reduced cell swelling in the resting zone and increased cell number by accelerating proliferation or inhibiting apoptosis. CNP thickened the proliferative and hypertrophic zones. Regarding changes in the bone, ERT restored bone sclerosis through decreased bone formation and increased bone resorption, and CNP did not adversely affect this process. In addition, improvement of joint deformation by ERT was suggested by analyses of joint spaces and articular cartilage. CNP additively provided restoration of the short stature of MPS VII mice in combination with ERT, which improved abnormalities of growth plates and bone metabolism.

First Report of a Patient with MPS Type VII, Due to Novel Mutations in GUSB, Who Underwent Enzyme Replacement and Then Hematopoietic Stem Cell Transplantation.

We report the case of a boy who was diagnosed with mucopolysaccharidosis (MPS) VII at two weeks of age. He harbored three missense ß-glucuronidase (GUSB) variations in exon 3: two novel, c.422A>C and c.424C>T, inherited from his mother, and the rather common c.526C>T, inherited from his father. Expression of these variations in transfected HEK293T cells demonstrated that the double mutation c.422A>C;424C>T reduces ß-glucuronidase enzyme activity. Enzyme replacement therapy (ERT), using UX003 (vestronidase alfa), was started at four months of age, followed by a hematopoietic stem cell allograft transplantation (HSCT) at 13 months of age. ERT was well tolerated and attenuated visceromegaly and skin infiltration. After a severe skin and gut graft-versus-host disease, ERT was stopped six months after HSCT. The last follow-up examination (at the age of four years) revealed a normal psychomotor development, stabilized growth curve, no hepatosplenomegaly, and no other organ involvement. Intriguingly, enzyme activity had normalized in leukocytes but remained low in plasma. This case report illustrates: (i) The need for an early diagnosis of MPS, and (ii) the possible benefit of a very early enzymatic and/or cellular therapy in this rare form of lysosomal storage disease.

Intrathecal AAVrh10 corrects biochemical and histological hallmarks of mucopolysaccharidosis VII mice and improves behavior and survival.

Mucopolysaccharidosis (MPS) type VII is a lysosomal storage disease caused by ß-glucuronidase deficiency, prompting glycosaminoglycan accumulation in enlarged vesicles, leading to peripheral and neuronal dysfunction. Here, we present a gene therapy strategy using lumbar puncture of AAVrh10 encoding human ß-glucuronidase (AAVrh10-GUSB) to adult MPS VII mice. This minimally invasive technique efficiently delivers the recombinant vector to the cerebrospinal fluid (CSF) with a single intrathecal injection. We show that AAVrh10 delivery to the CSF allows global, stable transduction of CNS structures. In addition, drainage of AAVrh10-GUSB from the CSF to the bloodstream resulted in the transduction of somatic organs such as liver, which provided a systemic ß-glucuronidase source sufficient to achieve serum enzyme activity comparable to wild type mice. ß-glucuronidase levels were enough to correct biochemical and histopathological hallmarks of the disease in the CNS and somatic organs at short and long term. Moreover, the progression of the bone pathology was also reduced. Importantly, the biochemical correction led to a significant improvement in the physical, cognitive and emotional characteristics of MPS VII mice, and doubling their life span. Our strategy may have implications for gene therapy in patients with lysosomal storage diseases.

A new case report of severe mucopolysaccharidosis type VII: diagnosis, treatment with haematopoietic cell transplantation and prenatal diagnosis in a second pregnancy.

A new patient with severe mucopolysaccharidosis (MPS) type VII is reported. Non-immune hydrops fetalis (NIHF) was diagnosed during pregnancy. At birth, he showed generalized hydrops and dysmorphic features typical of MPS. Many diagnoses were excluded before reaching the diagnosis of MPS VII at 8 months of life. During the first year of life he had frequent respiratory infections associated with restrictive and obstructive bronchopneumopathy and underwent three surgical interventions: decompression of the spinal cord at the craniocervical junction, bilateral inguinal hernia, and bilateral clubfoot. At 14 months of life he underwent successful haematopoietic cell transplantation (HCT). During the following 10 months, his bronchopneumopathy progressively worsened, needing chronic pharmacological treatment and O2 administration. The patient died of respiratory insufficiency during a respiratory syncytial virus infection at 25 months of age. Molecular analysis showed the homozygous variant c.1617C > T, leading to the synonymous mutation p.Ser539=. This caused aberrant splicing with partial skipping of exon 10 (r.1616_1653del38) and complete skipping of exon 9 (r.1392_1476del85; r.1616_1653del38). No transcript of normal size was evident. The parents were both confirmed to be carriers. In a subsequent pregnancy, a prenatal diagnosis showed an affected fetus. Ultrasound examination before abortion showed NIHF. The skin and placenta examination by electron microscopy showed foamy intracytoplasmic vacuoles with a weakly electron-dense substrate. MPS VII is a very rare disease but it is possible that some cases go undiagnosed for several reasons, including that MPS VII, and other lysosomal storage diseases, are not included in the work-up for NIHF in many institutions, and the presence of anasarca at birth may be confounding for the recognition of the typical facial characteristics of the disease. This is the eighth patient affected by MPS VII who has undergone HCT. It is not possible to draw conclusions about the efficacy of HCT in MPS VII. Treatment with enzyme replacement is now available and will probably be beneficial for the patients who have a milder form with no or little cognitive involvement. Increased awareness among clinicians is needed for prompt diagnosis and to offer the correct treatment as early as possible.

Haematopoietic stem cell transplantation for mucopolysaccharidosis type VII: A case report.

Mucopolysaccharidosis type VII (MPS VII) is an inherited disease characterized by the cellular accumulation of undegraded GAGs due to the deficiency of the lysosomal enzyme ß-glucuronidase. We describe a case of a 2-year-old female affected by a moderate form of MPS VII and submitted twice to HSCT with the aim of stabilizing skeletal problems and preventing neurocognitive alterations. The child underwent a second transplantation due to the rejection of the graft after a reduced-intensity conditioning in the first transplant. A myeloablative regimen allowed to achieve a stable full donor engraftment and normal enzyme levels during the 6 years of follow-up. Clinically, we observed stabilization of skeletal deformities and normal neurocognitive development. This is one of the few reports of mucopolysaccharidosis type VII treated with allogeneic HSCT.

A novel Blind Start study design to investigate vestronidase alfa for mucopolysaccharidosis VII, an ultra-rare genetic disease.

BACKGROUND: Drug development for ultra-rare diseases is challenging because small sample sizes and heterogeneous study populations hamper the ability of randomized, placebo-controlled trials with a single primary endpoint to demonstrate valid treatment effects. METHODS: To overcome these challenges, a novel Blind Start design was utilized in a study of vestronidase alfa in mucopolysaccharidosis VII (Sly syndrome), an ultra-rare lysosomal disease, that demonstrates the strengths of this approach in a challenging drug-development setting. Twelve subjects were randomized to 1 of 4 blinded groups, each crossing over to active treatment in a blinded fashion at different timepoints with efficacy analysis comparing the last assessment before cross over to after 24 weeks of treatment. Study assessments included: Percentage change from baseline in urinary GAG (uGAG); a Multi-Domain Responder Index (MDRI) using prespecified minimal important differences (6-Minute Walk Test, Forced Vital Capacity, shoulder flexion, visual acuity, and Bruininks-Oseretsky Test of Motor Proficiency); fatigue as assessed by the Pediatric Quality of Life Inventory™ Multidimensional Fatigue Scale; and safety. RESULTS: Vestronidase alfa treatment for 24 weeks significantly reduced uGAG excretion (dermatan sulfate: 64.8%, p < 0.0001). Most subjects (10/12) had a clinically meaningful improvement in at least one MDRI domain with an overall mean change (±SD) of +0.5 (±0.8) at Treatment Week 24 (p = 0.0527). Exposure-adjusted incidence rates of adverse events were similar between groups. CONCLUSIONS: The Blind Start study and MDRI design improve statistical power that enhances detection of a positive treatment effect in this rare heterogeneous disease and could be utilized for other ultra-rare diseases.

Reversal of established bone pathology in MPS VII mice following lentiviral-mediated gene therapy.

Severe, progressive skeletal dysplasia is a major symptom of multiple mucopolysaccharidoses (MPS) types. While a gene therapy approach initiated at birth has been shown to prevent the development of bone pathology in different animal models of MPS, the capacity to correct developed bone disease is unknown. In this study, ex vivo micro-computed tomography was used to demonstrate that bone mass and architecture of murine MPS VII L5 vertebrae were within the normal range at 1month of age but by 2months of age were significantly different to normal. The difference between normal and MPS VII BV/TV increased with age reaching a maximal difference at approximately 4months of age. In mature MPS VII bone BV/TV is increased (51.5% versus 21.5% in normal mice) due to an increase in trabecular number (6.2permm versus 3.8permm in normal mice). The total number of osteoclasts in the metaphysis of MPS VII mice was decreased, as was the percentage of osteoclasts attached to bone. MPS VII osteoblasts produced significantly more osteoprotegerin (OPG) than normal osteoblasts and supported the production of fewer osteoclasts from spleen precursor cells than normal osteoblasts in a co-culture system. In contrast, the formation of osteoclasts from MPS VII spleen monocytes was similar to normal in vitro, when exogenous RANKL and m-CSF was added to the culture medium. Administration of murine ß-glucuronidase to MPS VII mice at 4months of age, when bone disease was fully manifested, using lentiviral gene delivery resulted in a doubling of osteoclast numbers and a significant increase in attachment capacity (68% versus 29.4% in untreated MPS VII animals). Bone mineral volume rapidly decreased by 39% after gene therapy and fell within the normal range by 6months of age. Collectively, these results indicate that lentiviral-mediated gene therapy is effective in reversing established skeletal pathology in murine MPS VII.

Evaluation of AAV-mediated Gene Therapy for Central Nervous System Disease in Canine Mucopolysaccharidosis VII.

Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease arising from mutations in ß-d-glucuronidase (GUSB), which results in glycosaminoglycan (GAG) accumulation and a variety of clinical manifestations including neurological disease. Herein, MPS VII dogs were injected intravenously (i.v.) and/or intrathecally (i.t.) via the cisterna magna with AAV9 or AAVrh10 vectors carrying the canine GUSB cDNA. Although i.v. injection alone at 3 days of age resulted in normal cerebrospinal fluid (CSF) GUSB activity, brain tissue homogenates had only ~1 to 6% normal GUSB activity and continued to have elevated GAG storage. In contrast, i.t. injection at 3 weeks of age resulted in CSF GUSB activity 44-fold normal while brain tissue homogenates had >100% normal GUSB activity and reduced GAGs compared with untreated dogs. Markers for secondary storage and inflammation were eliminated in i.t.-treated dogs and reduced in i.v.-treated dogs compared with untreated dogs. Given that i.t.-treated dogs expressed higher levels of GUSB in the CNS tissues compared to those treated i.v., we conclude that i.t. injection of AAV9 or AAVrh10 vectors is more effective than i.v. injection alone in the large animal model of MPS VII.

Ex vivo gene therapy using patient iPSC-derived NSCs reverses pathology in the brain of a homologous mouse model.

Neural stem cell (NSC) transplantation is a promising strategy for delivering therapeutic proteins in the brain. We evaluated a complete process of ex vivo gene therapy using human induced pluripotent stem cell (iPSC)-derived NSC transplants in a well-characterized mouse model of a human lysosomal storage disease, Sly disease. Human Sly disease fibroblasts were reprogrammed into iPSCs, differentiated into a stable and expandable population of NSCs, genetically corrected with a transposon vector, and assessed for engraftment in NOD/SCID mice. Following neonatal intraventricular transplantation, the NSCs engraft along the rostrocaudal axis of the CNS primarily within white matter tracts and survive for at least 4 months. Genetically corrected iPSC-NSCs transplanted post-symptomatically into the striatum of adult Sly disease mice reversed neuropathology in a zone surrounding the grafts, while control mock-corrected grafts did not. The results demonstrate the potential for ex vivo gene therapy in the brain using human NSCs from autologous, non-neural tissues.

The effect of Tlr4 and/or C3 deficiency and of neonatal gene therapy on skeletal disease in mucopolysaccharidosis VII mice.

Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder caused by the deficiency of the enzyme ß-glucuronidase (Gusb(-/-)) and results in glycosaminoglycan (GAG) accumulation. Skeletal abnormalities include stunted long bones and bone degeneration. GAGs have been hypothesized to activate toll-like receptor 4 (Tlr4) signaling and the complement pathway, resulting in upregulation of inflammatory cytokines that suppress growth and cause degeneration of the bone. Gusb(-/-) mice were bred with Tlr4- and complement component 3 (C3)-deficient mice, and the skeletal manifestations of the doubly- and triply-deficient mice were compared to those of purebred Gusb(-/-) mice. Radiographs showed that purebred Gusb(-/-) mice had shorter tibias and femurs, and wider femurs, compared to normal mice. No improvement was seen in Tlr4, C3, or Tlr4/C3-deficient Gusb(-/-) mice. The glenoid cavity and humerus were scored on a scale from 0 (normal) to +3 (severely abnormal) for dysplasia and bone irregularities, and the joint space was measured. No improvement was seen in Tlr4, C3, or Tlr4/C3-deficient Gusb(-/-) mice, and their joint space remained abnormally wide. Gusb(-/-) mice treated neonatally with an intravenous retroviral vector (RV) had thinner femurs, longer legs, and a narrowed joint space compared with untreated purebred Gusb(-/-) mice, but no improvement in glenohumeral degeneration. We conclude that Tlr4- and/or C3-deficiency fail to ameliorate skeletal abnormalities, and other pathways may be involved. RV treatment improves some but not all aspects of bone disease. Radiographs may be an efficient method for future evaluation, as they readily show glenohumeral joint abnormalities.

Partial rescue of mucopolysaccharidosis type VII mice with a lifelong engraftment of allogeneic stem cells in utero.

In utero hematopoietic cell transplantation (IUHCT) has been performed in Mucopolysaccharidosis Type VII (MPSVII) mice, but a lifelong engraftment of allogeneic donor cells has not been achieved. In this study, we sought to confirm a lifelong engraftment of allogeneic donor cells immunologically matched to the mother and to achieve partial rescue of phenotypes in the original MPSVII strain through IUHCT by intravenous injection. We performed in vitro fertilization in a MPSVII murine model and transferred affected embryos to ICR/B6-GFP surrogate mothers in cases where fetuses receiving IUHCT were all homozygous. Lineage-depleted cells from ICR/B6-GFP mice were injected intravenously at E14.5. Chimerism was confirmed by flow cytometry at 4 weeks after birth, and ß-glucuronidase activity in serum and several phenotypes were assessed at 8 weeks of age or later. Donor cells in chimeric mice from ICR/B6-GFP mothers were detected at death, and were confirmed in several tissues including the brains of sacrificed chimeric mice. Although the serum enzyme activity of chimeric mice was extremely low, the engraftment rate of donor cells correlated with enzyme activity. Furthermore, improvement of bone structure and rescue of reproductive ability were confirmed in our limited preclinical study. We confirmed the lifelong engraftment of donor cells in an original immunocompetent MPSVII murine model using intravenous IUHCT with cells immunologically matched to the mother without myeloablation, and the improvement of several phenotypes.

Mucopolysaccharidosis type VII: A powerful experimental system and therapeutic challenge.

Mucopolysaccharidosis type VII (MPSVII) is an inborn error of metabolism caused by a deficiency in the lysosomal enzyme B-glucuronidase (GUSB). As such, MPSVII is one of a larger class of inherited diseases referred to as lysosomal storage diseases (LSD). (1) The absence of GUSB activity leads to the progressive accumulation of undegraded glycosaminoglycans (GAGs) in many tissues of the body. Mucopolysaccharidosis VII has a complex clinical phenotype, including skeletal dysplasia, hepatosplenomegally, sensory deficits, cognitive impairment, and premature death. Although the natural history of the human disease is not precisely defined, small and large animal models of MPSVII have played a major role in our understanding of the disease process and towards effective treatments. The mouse model of MPSVII is a particularly powerful system due to its similarity to the human disease and the ability to generate large numbers of genetically defined animals. It has been shown in the murine model of MPSVII that recombinant enzyme replacement therapy (ERT) can ameliorate most of the clinical signs of disease if initiated during the neonatal period. Progenitor cell transplantation (hematopoietic, neuronal, mesenchymal) can correct many of the pathological signs of disease in MPSVII mice. Viral-mediated gene therapy has also been shown to decrease the severity of the disease in both the murine and canine models of MPSVII. Although pre-clinical experiments have shown that a number of approaches can effectively treat MPSVII, translation of those therapies into the clinic has lagged behind other LSDs. This is due in large part to the ultra-rare nature of MPSVII. Encouragingly, a clinical trial of ERT for MPSVII has recently been initiated. It will be interesting to determine if the positive pre-clinical data gathered in animal models of MPSVII translate to affected children. This clinical trial may also establish a paradigm for the treatment of other ultra-rare disorders.