Shire Human Genetic Therapies Research Articles

Nrf2 Null Mice Are Deficient in CD169+ Macrophages, Associated with Impaired Erythroid Response and Delayed Recovery from Acute Blood Loss

Erythropoiesis occurs in specialized niches in the bone marrow consisting of a central macrophage, surrounded by differentiating erythroblasts. This central macrophage has been identified by several markers including, CD169 (Sialoadhesin or Siglec-1), F4/80, CD11b, VCAM-1, ER-HR3 and Ly-6G. These CD169+ macrophages support erythropoiesis both at steady state and during stress. Nuclear factor erythroid 2-related factor 2 (Nrf2) is the master regulator of the cellular oxidative defense system. It modulates hematopoietic stem cells but its loss produces no visible phenotype in steady state hematological parameters. However, the importance of Nrf2 and macrophage subsets has not been fully characterized during recovery from stress erythropoiesis. We examined specific subsets of CD169+ macrophage populations in Nrf2 knockout (Nrf2−/−) mice as well as the role of Nrf2 in recovery from stress erythropoiesis in vivo. We quantified the expression of CD169, F4/80 and CD11b, markers of central macrophages, in the BM and spleen of Nrf2+/+ and Nrf2-/- mice at steady state. Surprisingly, Nrf2-/- mice showed a phenotype characterized by lower percentages of cells expressing known macrophage markers. We observed a significant decrease of 47% (p≤0.01), 24% (p≤0.01) and 50% (p≤0.01) in BM macrophage subpopulations expressing F4/80hiCD169hi, F4/80hiCD11bhi and CD169hiCD11bhi respectively, in age-matched Nrf2-/- mice compared to Nrf2+/+ control mice (Fig. 1a). In the spleen, we also observed a similar significant deficiency in BM macrophages (p≤0.01). Further validating this phenotype, immunofluorescence staining of isolated spleen tissue showed that expression of CD169+ macrophages was dramatically lower in spleen sections of Nrf2-/- mice than in Nrf2+/+ control mice. We hypothesized that our macrophage-deficient mice would display a defect in recovery from blood loss. Five to seven days after acute blood loss, immature erythroid progenitors (CD71hiTer119hi) increased in marrow by about 5-fold in Nrf2+/+ mice (p≤0.001, Fig. 1b), and mature erythroid progenitors (CD71loTer119hi) increased in marrow by 12-fold (p≤0.05) but the erythroid marrow response was impaired significantly in the macrophage deficient Nrf2-/- mice. To extend our observations regarding macrophage deficiency and impaired erythroid response, we chose a more functional outcome of recovery from anemia after high-grade blood loss produced by daily phlebotomies over 3 consecutive days to induce stress erythropoiesis in Nrf2+/+ and Nrf2-/- mice. We found significantly lower packed cell volume values specifically on Days 2, 4 and 10, implying delayed erythroid recovery (p≤0.05, two-way ANOVA). The Nrf2-/- mice also showed a significant decline in total hemoglobin than the Nrf2+/+ mice (p≤0.05). Additionally, peripheral blood reticulocyte response to blood loss is delayed in Nrf2 deficient mice compared to age-matched controls (11.0 ± 0.6% vs. 14.8 ± 0.6%, p≤0.001). We analyzed expression of heme-oxygenase 1 (HO-1), a well-known Nrf2-regulated gene. HO-1 mRNA expression increased 3-fold and 23-fold in Nrf2+/+ mice animals subjected to phlebotomy and hemin treatment compared to 2-fold and 12-fold expression in Nrf2-deficient mice (p≤0.05). We demonstrate for the first time that Nrf2-deficient mice have a deficiency of macrophages that includes subsets considered erythroblastic island (EI) macrophages, and that this deficiency is associated with impaired erythroid response to induced stress. Secondly, our multiple phlebotomies data in aggregate demonstrate that Nrf2-/- mice deficient in BM macrophages have significant delay in functional erythroid response and recovery from experimentally-induced anemia. Thirdly, impaired inducibility of HO-1 is a known feature of Nrf2-/- mice, which we confirmed in our results, could be contributing to the impairment in erythroid response. However, it is not likely that restricted iron trafficking to erythroid progenitors occurs in Nrf2-/- mice, since there is no characteristic alteration of mean corpuscular volume and mean corpuscular hemoglobin in peripheral blood. This is an area worthy of additional investigation. We conclude that the Nrf2 gene plays a previously unappreciated role in erythroid biology that appears to be mediated through macrophage function. Disclosures Ofori-Acquah: Shire Human Genetic Therapies Inc: Other: Financial Relationship. Kato:Bayer: Research Funding; Novartis, Global Blood Therapeutics: Consultancy, Research Funding.

Macrophage Hypercellularity Accompanies Erythroid Hyperplasia in Sickle Cell Mice and during Recovery from Blood Loss in Wild Type Mice

Red blood cell (RBC) homeostasis tightly balances production of new erythroid cells and clearance of damaged or senescent RBCs. Macrophages play a central role in this process in the specialized niches termed erythroblastic island. Macrophages expressing CD169 (Sialoadhesin or Siglec-1), F4/80, CD11b, VCAM-1, ER-HR3 and Ly-6G play important roles in physiological and pathological erythropoiesis. Sickle cell disease (SCD) is characterized by chronic stress erythropoiesis as a compensatory mechanism for anemia. Recent publications have detected macrophage hypercellularity correlated with erythroid hyperplasia in genetic models of erythrocytosis and monocyte-derived macrophage proliferation in the spleen. Because macrophages are integral part of the erythropoietic niche, our objective was to assess the macrophage compartment in transgenic sickle cell mice (SS) at steady state compared to AA controls. Furthermore, we used acute blood loss in C57BL/6J wild type (WT) to confirm if macrophage hypercellularity accompanies erythroid hyperplasia in a mouse model without erythrocytosis or sickling. Using flow cytometry, macrophages were identified with F4/80, CD45, CD11b and CD169, while erythroid progenitor cells were identified with Ter119 and CD71 in the BM and spleen of SS mice and age-matched control (AA) mice. Each subset of macrophages is 3.8 to 6.8-fold higher in SS mice bone marrow compared to AA at steady state (p≤0.05, Fig 1a). Findings were similar for other CD169 subsets in the spleens of SS mice. This macrophage hypercellularity was accompanied by expected erythroid hyperplasia, with 12-fold higher immature erythroid progenitor cells (CD71hiTer119hi) in SS mice than AA controls (p≤0.001). To confirm the increase in macrophage numbers that accompanied erythroid hyperplasia in SS mice as a response to erythroid stress, we induce stress with acute blood loss in WT mice by phlebotomy (once). Interestingly, we found macrophage hypercellularity accompanies erythroid hyperplasia in WT mice during recovery from acute blood loss. WT mice showed about 47-61% increases in CD169 macrophages subsets in the marrow and 47-83% increase in the spleen during recovery from acute blood loss (5-7 days) compared to untreated mice (Fig. 1b). This macrophage hypercellularity was accompanied with 88% increase in immature erythroid progenitor cell. Similarly, we investigated if there was age dependence in macrophage hypercellularity that accompanies erythroid hyperplasia in SS mice. In SS mice, there was no significant differences in macrophage numbers in the BM juvenile mice (5 weeks old) compared to young adults (14 weeks old), presumably because the higher demand for erythroid production to compensate for anemia had already started in the juvenile animals. However, there was a significantly higher macrophage hypercellularity in the spleen at 14 weeks compared to juvenile animals (p≤0.001). There was also a reduction in hematocrit (p≤0.05) and hemoglobin (p≤0.01) from 5 to 14 weeks in SS mice, although this does not correlate to either macrophage hypercellularity or erythroid hyperplasia. We also quantified mRNA expression of heme oxygenase 1 (Hmox1), a critical regulator of erythroid stress response, in the BM of untreated SS mice, and untreated and stressed WT mice. As expected, BM cells from SS mice at steady state expressed significantly higher Hmox1 expression than in WT mice (p≤0.001). Acute blood loss induced-stress raised the Hmox1 expression in WT mice to comparable levels to that of SS mice at steady state. Our investigation is the first to our knowledge to detect BM macrophage hypercellularity is associated with erythroid hyperplasia in response to acute blood loss and in congenital hemolytic anemia. Our data of macrophage hypercellularity in SCD opens up further research opportunity to elucidate the role of these cells in multiorgan complication in SCD. Figure 1: Macrophage hypercellularity accompanies erythroid hyperplasia in sickle cell disease and during recovery from acute blood loss. (a) BM macrophage quantification (per femur) in untreated sickle cell mice compared to AA controls (n = 5). Student's Ttest * SS mice compared to AA mice. (b) Acute blood loss induced macrophage hypercellularity in the BM of WT mice (n = 4 - 5). Student's Ttest * Untreated compared to stressed WT mice. Figure 1 Disclosures Ofori-Acquah: Shire Human Genetic Therapies Inc: Other: Financial Relationship. Kato:Novartis, Global Blood Therapeutics: Consultancy, Research Funding; Bayer: Research Funding.

Hemopexin Replacement Therapy Protects Sickle Cell Disease Mice from Acute Kidney Injury

Acute kidney injury (AKI) is a major clinical concern during episodes of acute chest syndrome and vaso-occlusive crisis in sickle cell disease (SCD). AKI increases the risk of chronic kidney disease (CKD) and end stage renal disease (ESRD). We previously showed that alpha-1-microglobulin (A1M), a low affinity heme-binding protein that carries heme for renal clearance is elevated in patients and mice with homozygous SCD (SS). Hemopexin (Hx), which primarily scavenges circulating heme to the liver, is exhausted in SCD. In this study, we explored the idea that acquired Hx deficiency in SCD is a risk factor for AKI development in SCD. We studied mice with a global knockout of Hx with no detectable plasma Hx at baseline. Plasma A1M was significantly elevated in the Hx-/-mice compared to control (Hx+/+) mice (n=5; p<0.01). We then transplanted whole bone marrow cells from SS mice into Hx+/+and Hx-/-mice to create bone marrow chimeric SSHx+/+and SSHx-/-mice with SCD phenotype. The chimeras had elevated plasma A1M compared to recipient littermates (Hx+/+and Hx-/-) and low baseline glomerular filtration rate (GFR). Modest elevation of circulating heme with infusion of hemin (20 μmoles/kg bw) worsened GFR and caused severe AKI. Next, to determine whether hemin induced AKI is attenuated by elevation of circulating Hx, we infused SS mice with purified Hx, and control SS mice with either purified A1M or vehicle immediately prior to the hemin challenge. We observed improved GFR in the Hx-treated SS mice, while vehicle and A1M-treated mice suffered 23% and 39% loss of GFR respectively compared to their baseline. In agreement with the GFR data, the levels of several AKI diagnostic markers, plasma creatinine (plasma Cr), urinary albumin-creatinine ratio (uACR) and kidney injury molecule (uKIM-1) were elevated significantly in vehicle and A1M treated mice following hemin challenge. In Hx-treated mice, these biomarkers remained unaltered. Importantly, Hx infusions re-directed excess heme in SS mice to the liver, while A1M infusion significantly increased total heme content in the kidneys. Two-way ANOVA analysis of GFR (p<0.01) and plasma Cr (p<0.001) revealed significant exacerbation of kidney injury in A1M treated SS mice compared to vehicle treated mice. Histopathology of renal tissue showed considerable tissue damage in vehicle and A1M infused SS mice, while Hx treated SS mice kidneys appeared relatively normal. This study provides genetic evidence that hemopexin deficiency promotes AKI development in SCD, and we provide proof-of-principle for hemopexin replacement therapy to treat AKI in SCD. Disclosures Ofori-Acquah: Shire Human Genetic Therapies Inc: Other: Financial Relationship.

Smooth Muscle Cytochrome b5 Reductase 3 Deficiency Accelerates Pulmonary Hypertension Development in Sickle Cell Mice

Pulmonary and systemic vasculopathy are significant risk factors for early morbidity and death in patients with sickle cell disease (SCD). An underlying mechanism of SCD vasculopathy is vascular smooth muscle (VSM) nitric oxide (NO) resistance, which is mediated by NO scavenging reactions with plasma hemoglobin and reactive oxygen species that can oxidize soluble guanylyl cyclase (sGC), the NO receptor. Prior studies show that cytochrome b5 reductase 3 (CYB5R3), known as methemoglobin reductase in erythrocytes, functions in VSM as an sGC heme iron reductase critical for reducing and sensitizing sGC to NO and generating cGMP for vasodilation. Therefore, we hypothesized that VSM CYB5R3 deficiency accelerates development of pulmonary hypertension (PH) in SCD. Bone marrow transplant was used to create SCD chimeric mice with background smooth muscle cell (SMC) specific tamoxifen-inducible Cyb5r3 knockout (SMC R3 KO) and wild-type (WT) control.Three weeks after completing tamoxifen treatment, we observed 60% knockdown of pulmonary arterial SMC CYB5R3, 5-6 mmHg elevated right ventricular maximum systolic pressure (RVmaxSP) and biventricular hypertrophy in SS chimeras with SMC R3 KO (SS/R3KD) relative to wild-type (SS/R3WT). Right ventricular (RV) contractility, heart rate, hematological parameters and cell-free hemoglobin were similar between groups. When identically generated SS/R3 chimeras were studied 12-weeks after completing tamoxifen treatment, RVmaxSP in SS/R3KD had not increased further, but RV hypertrophy relative to SS/R3WT persisted. These are the first studies to establish involvement of SMC CYB5R3 in SCD-associated development of PH, which can exist in mice by 5 weeks of SMC CYB5R3 protein deficiency. Disclosures Vitturi: Complexa Inc: Consultancy. Ofori-Acquah:Shire Human Genetic Therapies Inc: Other: Financial Relationship. Gladwin:Globin Solutions, Inc: Patents & Royalties: Provisional patents for the use of recombinant neuroglobin and heme-based molecules as antidotes for CO poisoning; Bayer Pharmaceuticals: Other: Co-investigator; United Therapeutics: Patents & Royalties: Co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases . Straub:Bayer Pharmaceuticals: Research Funding.

Targeted Therapy of Acute Chest Syndrome with Recombinant Heme Oxygenase-1 Revealed By the Resistance of Young Transgenic Sickle Cell Mice to Heme-Induced Acute Lung Injury

Acute chest syndrome (ACS) is a major pulmonary complication of sickle cell disease (SCD) with currently no mechanistic-based therapies. Although ACS is more frequent in childhood, survival among children 1-9 years is ~10-fold higher compared to adults 20 years and older. Hitherto, mechanisms responsible for the high ACS survival in children have not been defined. We previously reported that acute elevation of circulating heme causes a TLR4-dependant lethal acute lung injury (ALI) in transgenic mice with homozygous SCD (SS) reminiscent of severe ACS (Ghosh et al., J Clin Invest, 2013). In the current study we discovered that young (4-6 weeks) and adult (12 weeks) SS mice suffer comparable severity of hemolytic crises when challenged with intravenous hemin (35 μmoles/kg bw). However, virtually none of the young mice develop ALI while nearly all the adults succumb with respiratory failure. We discovered that young SS mice rapidly clear excess heme from the circulation suggesting this was the reason for their resistance to intravenous hemin. Interestingly, young SS mice have significantly lower plasma hemopexin levels than adult SS mice, which excludes the classical heme scavenging pathway as the reason for their heme resistance. Previous studies have linked genetically high heme oxygenase-1 (HO-1) expression with low-risk of ACS, and raised steady-state levels of plasma heme with high-risk ACS in children. To explore these associations further, we studied a large cohort of patients and discovered for the first time that the concentration of plasma HO-1 in SCD children 1-9 yrs is 2-fold higher than adults 20 yrs and older (23.6±1.1, n=191 versus mean 10.7±0.6, n=67); these findings were phenocopied in transgenic SS mice. Plasma fractionization experiments revealed that HO-1 co-localizes with the enzymatic and co-factor machinery required for heme degradation indicating that heme can be degraded in the blood circulation. To test this idea ~3 weeks old SS mice were treated with the HO-1 inhibitor tin protoporphyrin (SnPP) or vehicle, and challenged with intravenous hemin five days later to induce ALI/ACS. Plasma HO activity declined by ~76% in SnPP-treated mice and was unchanged in the vehicle treated animals (n=12-14, p<0.001). A significant majority (10/12; 83%) of the vehicle-treated mice survived, while a majority of the SnPP-treated mice (10/14; 71%) developed lethal ALI/ACS. In another series of experiments young SS mice were treated for three months with Nrf2 activator to block age-dependent HO-1 decline, and then treated with vehicle or SnPP prior to ALI/ACS induction. The SnPP-treated group had 58% reduction in plasma HO-1 (p<0.05), failed to clear heme and developed lethal ALI/ACS, while a majority (63%) of the vehicle treated SS mice with persistently raised HO-1 activity in adulthood survived. Finally, adult SS mice were treated acutely with novel truncated human HO-1 recombinant proteins or a carrier vehicle immediately prior to induction of ALI/ACS. Recombinant variant 4 (V4) possessing an Fc component that enhances protein half-life provided 100% protection while 70% of vehicle treated mice succumbed with ALI/ACS (Mantel-Cox log-rank p<0.001). While the arterial oxygen saturation level (% SpO2) in vehicle treated SS mice declined sharply reflecting severe respiratory distress, V4-treated mice maintained normal SpO2 (p<0.001). In conclusion, we have determined that HO-1 activity in blood plasma declines with aging in SCD patients and identify this phenomenon as the basis of poor survival among adult SS mice with induced ALI/ACS. Furthermore, we provide proof-of-principle in mice that a novel human recombinant HO-1 protein can attenuate lung injury and improve survival from ALI/ACS in SCD. Disclosures Ofori-Acquah: Shire Human Genetic Therapies Inc: Other: Financial Relationship.

Predictors of Response to Teduglutide Among Patients with Parenteral Nutrition–Dependent Short Bowel Syndrome

Introduction: The study aimed to identify factors associated with sustained parenteral nutrition (PN) volume reduction and early vs late responders among PN-dependent short bowel syndrome patients treated with teduglutide (TED). Methods: The study population was 43 patients receiving TED in a 24-week randomized placebo-controlled trial (STEPS: NCT00798967; EudraCT2008-006193-15) who could continue TED up to 24 months in the open-label extension (STEPS-2: NCT00930644; EudraCT2009-011679-65). Time to sustained PN volume reduction (i.e., PN volume reduction ≥20% at 2 consecutive visits) was calculated using Kaplan-Meier analysis. Multivariable Cox model was used to identify factors associated with sustained PN volume reduction. Among patients achieving sustained PN volume reduction, baseline characteristics were compared between early (PN volume reduction ≥20% at week 20 and 24) vs. late responders (sustained PN volume reduction at 2 consecutive visits after week 24). Results: The median time to sustained PN volume reduction was 4.2 months. Patients with stoma, compared to those without, were significantly more likely to achieve sustained PN volume reduction (hazard ratio [HR], 5.6; P = 0.01). Patients with an ileocecal valve, compared to those without (HR = 0.1; P = 0.03), and patients with major intestinal resection due to vascular disease, compared to those with Crohn’s disease (HR = 0.2; P = 0.02), were significantly less likely to achieve sustained PN volume reduction. Compared to late responders (n = 7), early responders (n = 27) were less likely to have colon-in-continuity (52% vs 100%; P = 0.02) and ileocecal valve (0% vs 29%; P < 0.01), and had smaller mean percentage of colon remaining (25% vs 57%; P = 0.02). The median time to sustained PN volume reduction was 3.7 months for early responders and 7.8 months for late responders. Conclusion: Presence of stoma and absence of ileocecal valve are good prognosis factors for achieving sustained PN volume reduction while vascular disease as cause of intestinal resection is a negative prognosis factor. Certain anatomical factors were significantly different between early and late responders. This research was funded by Shire Human Genetic Therapies, Inc., Lexington, MA, USA.

Velaglucerase alfa in the treatment of Gaucher disease type 1: an update

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by deficiency of the enzyme acid β-glucosidase. Enzyme replacement therapy is the standard of care for the treatment of GD type I. Currently, three preparations, including imiglucerase (Cerezyme®, Genzyme Corporation, MA, USA), taliglucerase alfa (Elelyso®, Pfizer Inc., NY, USA) and velaglucerase alfa (VPRIV®, Shire Human Genetic Therapies Inc., Dublin, Ireland), are commercially available. Here, we will review the recent literature addressing the safety and efficacy of velaglucerase, particularly as compared with the other enzyme replacement therapy products, as well as the treatment of GD type 1 with velaglucerase alfa.

Persistence of Imiglucerase in Cell Compartment Explains Dosing Frequency of Every Two Weeks in Type 1 Gaucher Disease

GD is a genetic lysosomal disease characterized by deficiency in glucocerebrosidase (Glc) particularly affecting macrophage-like cells where glucocerebroside accumulates in the lysosomes. Alglucerase then imiglucerase (IMI, Genzyme-Sanofi Corporation), a recombinant human glucocerebrosidase, improved or reversed many type 1 GD symptoms; Two new biosimilar agents are now available: velaglucerase-alfa (Shire Human Genetic Therapies) and taliglucerase-alfa (Biotherapeutics, Pfizer). All three recombinant enzymes are administered with identical therapeutic regimens, i.e. one infusion (30-60 IU/kg) every two weeks. However, disease responses remain variable and there are no pharmacokinetic explanations justifying this dosage regimen since plasma half-life is estimated to be less than 10 min. in murine models and in humans We compared endogenous glucocerebrosidase (Glc) activity evaluated by flow cytometry in the blood monocyte (Mo) compartment of 20 patients with 71 healthy donors, and we then followed residual intra-Mo Glc activity in patients starting imiglucerase (n=8).By using the only technique able to identify blood leukocyte subsets, we confirmed the marked higher value in Mo (×43) as compared to lymphocytes and polymorphonuclear (PMN) cells. In the 20 type 1 GD patients, we confirmed that enzyme deficiency was clear in Mo (p = 10-11 compared to HD), residual Glc activity in Mo from type 1 GD representing only 10% of normal Glc activity.We then followed trough Glc activity in 8 patients starting imiglucerase treatment de novo (n=1) or restarting imiglucerase after shortage (n=7), at 40 (n=1) or 60 U (n=7) /Kg/2 weeks. We observed progressive uptake over 6 months and a significant cumulative persistence of enzyme activity in monocyte compartment even 14 days after infusion.The estimated increase of residual intra-monocyte enzyme activity at M6 was 1.4 of endogenous level (p=.04) leading to a steady state value. The estimated rate constant of increase corresponded to an initial half-life of 2.8 days. These results explain for the first time the clinical efficacy of the routine infusion regimen, since intra-cellular pharmacokinetics are longer than plasma pharmacokinetics.Furthermore, we can hypothesize that intra-cellular pharmacokinetics after infusion may vary between patients, as suggested by the different peaks of activity and variable residual levels of enzyme activity observed in this study. In this limited series, we observed several profiles: i) Four patients ( #1,2,3, 8) of whom one received 40 U/kg/2 weeks, showed progressive Glc uptake correlated with improvement of clinical and biological parameters; this corresponds to patients with hematological phenotype only (Figure S1 A). ii) Other patients showed variability of Glc uptake (Figure S1 B); these patients were more heterogenous, with bone disease (n=2), splenectomy (n=2), or BM disorders (myelodysplasia, n=1). Consequently intra-Mo imiglucerase uptake appears to be variable, suggesting for the first time a possible relation between Glc activity in cell compartments, phenotype and clinical efficacy.In summary, the persistence of imiglucerase in monocyte compartments explains the efficacy of 2-week infusion rates and variability may be a factor of ERT efficiency DisclosuresStirnemann:Genzyme/Sanofi: Consultancy. Belmatoug:Genzyme/Sanofi and Shire: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Rose:Genzyme: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Berger:Genzyme/Sanofi and Shire: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

A biochemical and physicochemical comparison of two recombinant enzymes used for enzyme replacement therapies of hunter syndrome.

Mucopolysaccharidosis II (MPS II, Hunter syndrome; OMIM 309900) is an X-linked lysosomal storage disease caused by a deficiency in the enzyme iduronate-2-sulfatase (IDS), leading to accumulation of glycosaminoglycans (GAGs). For enzyme replacement therapy (ERT) of Hunter syndrome, two recombinant enzymes, idursulfase (Elaprase(®), Shire Human Genetic Therapies, Lexington, MA) and idursulfase beta (Hunterase(®), Green Cross Corporation, Yongin, Korea), are currently available in Korea. To compare the biochemical and physicochemical differences between idursulfase and idursulfase beta, we examined the formylglycine (FGly) content, specific enzyme activity, mannose-6-phosphate (M6P) content, sialic acid content, and in vitro cell uptake activity of normal human fibroblasts of these two enzymes.The FGly content, which determines the enzyme activity, of idursulfase beta was significantly higher than that of idursulfase (79.4 ± 0.9 vs. 68.1 ± 2.2 %, P < 0.001). In accordance with the FGly content, the specific enzyme activity of idursulfase beta was significantly higher than that of idursulfase (42.6 ± 1.1 vs. 27.8 ± 0.9 nmol/min/μg protein, P < 0.001). The levels of M6P and sialic acid were not significantly different (2.4 ± 0.1 vs 2.4 ± 0.3 mol/mol protein for M6P and 12.3 ± 0.7 vs. 12.4 ± 0.4 mol/mol protein for sialic acid). However, the cellular uptake activity of the normal human fibroblasts in vitro showed a significant difference (Kuptake, 5.09 ± 0.96 vs. 6.50 ± 1.28 nM protein, P = 0.017).In conclusion, idursulfase beta exhibited significantly higher specific enzyme activity than idursulfase, resulting from higher FGly content. These biochemical differences may be partly attributed to clinical efficacy. However, long-term clinical evaluations of Hunter syndrome patients treated with these two enzymes will be needed to demonstrate the clinical implications of significant difference of the enzyme activity and the FGly content.

Fabry disease: is there a role for enzyme replacement therapy?

Fabry disease: is there a role for enzyme replacement therapy?

Recessive Mutations in POLR3B Encoding the Second Largest Subunit of Pol III Cause a Rare Hypomyelinating Leukodystrophy (P05.136)

Objective: Search for mutations in the second largest subunit of RNA polymerase III (Pol III), POLR3B , in case negative for mutation in POLR3A . Background Leukodystrophies are a heterogeneous group of neurodegenerative disorders characterized by abnormal central nervous system white matter. It is estimated that at least 30% to 40% of patients with leukodystrophies remain without a precise diagnosis, despite extensive investigations. We recently demonstrated that mutations in POLR3A, encoding the largest subunit of RNA polymerase III (Pol III), were responsible for the majority of cases presenting with three clinically overlapping hypomyelinating leukodystrophy phenotypes. Design/Methods: We sequenced all exons, exon-intron boundaries and 39 and 59 UTR of POLR3B (NM_018082, hg19) on available genomic DNA from four 4H cases not found previously to carry POLR3A mutations. Results: We uncovered in four cases without POLR3A mutation, recessive mutations in POLR3B which codes for the second largest subunit of Pol III. All four cases were found to be compound heterozygote for mutations in POLR3B , with one missense mutation being common to all individuals. Conclusions: Mutations in genes coding for Pol III subunits are a major cause of childhood-onset hypomyelinating leukodystrophies typically characterized by prominent cerebellar dysfunction, oligodontia and hypogonadotropic hypogonadism. Supported by: Dr G Bernard received fellowship scholarships from the RMGA (Reseau de Medecine Genetique Appliquee) and FRSQ (Fonds de Recherche en Sante du Quebec). M. Tetreault received the Frederick Banting and Charles Best Doctoral scholarship from the CIHR (Canadian Institute of Health Research). This work was supported by Fondation sur les Leucodystrophies and the European Leukodystrophy Association (ELA). Disclosure: Dr. Tetreault has nothing to disclose. Dr. Choquet has nothing to disclose. Dr. Orcesi has nothing to disclose. Dr. Tonduti has nothing to disclose. Dr. Ballotin has nothing to disclose. Dr. Teichmann has nothing to disclose. Dr. Fribourg has nothing to disclose. Dr. Schiffmann has received personal compensation for activities with Amicus Therapeutics and Shire Human Genetic Therapies. Dr. Schiffmann has received research support from Shire Human Genetic Therapies, Amicus Therapeutics, and Genzyme Corporation. Dr. Brais has nothing to disclose. Dr. Vanderver has nothing to disclose. Dr. Bernard has received personal compensation for activities with Actelion Pharmaceuticals Canada Inc, Santhera, Venture in Research as a participant on advisory boards and research teams.

Clinical guidelines for diagnosing and managing ocular manifestations in children with mucopolysaccharidosis

The mucopolysaccharidoses (MPS) are a group of rare lysosomal storage disorders characterized by the accumulation of glycosaminoglycans in several tissues and organs. This accumulation results in an array of clinical manifestations and premature death in severe cases. Ocular problems are very common in children with MPS and may involve the cornea, sclera, trabecular meshwork, retina, optic nerve and also the posterior visual pathways. The aims of this study are to give an overview of ocular problems in MPS and to provide clinical guidelines for paediatric ophthalmologists for early diagnosis and management of ocular manifestations in children with MPS. Diagnostic problems may arise in children with severe corneal clouding, hampering visualization of the fundus. Intraocular pressures may be falsely high, even leading to suspicion and unnecessary pressure-lowering treatment. Simple interventions such as the use of prescription glasses or photochromatic glasses can considerably improve quality of life in children with MPS.

The Uptake of Recombinant Glucocerebrosidases by Blood Monocytes From Type 1 Gaucher Disease Patients Is Heterogeneous

Abstract 1102 Introduction:Gaucher disease (GD) is due to an inborn deficiency of glucocerebrosidase (GC), that leads to the accumulation of glucosylceramide in monocytes/macrophages, known as “Gaucher cells”, which are thought to be responsible for a wide range of symptoms. Imiglucerase (CEREZYME®, Genzyme Corporation) (IMI) is the first line treatment of type 1 GD patients. Two new biosimilar agents, velaglucerase-alfa (VPRIV®, Shire Human Genetic Therapies, Dublin, Ireland) (VEL) and taliglucerase-alfa (PROTALIX®, Biotherapeutics, Pfizer) (TAL), have been described as being similar to IMI but differing slightly in glycan structure which could have an impact on macrophage uptake and thus on therapeutic efficacy. However, the ability of native GD cells to capture recombinant enzymes (REs) remains unknown. We used blood monocytes (Mo) from GD patients and healthy donors (HD) as a model to compare the uptake of the three REs. Patients and methods:After informed consent was obtained, cells were obtained from healthy donor blood (n=34). The left over part of biological samples collected for routine analysis from Gaucher patients (n=6) could be used for research because patients had been informed and did not verbally express any disagreement. Mononuclear cells were incubated with three concentrations of each RE (0.1, 0.5 and 1 U/ml) at 37°C for 30 minutes and one hour and washed twice with PBS to eliminate exogenous RE before evaluation of intra-cellular GC activity by standardized flow cytometry as previously described (Berger J. et al., Br J Haematol 2010) Results:Firstly, we confirmed that GD Mo (n=6) showed a marked enzyme deficiency (about 7% of the normal endogenous activity) as compared to normal glucocerebrosidase activity (GCA) (n=34) Then we observed a dose-dependent in vitro uptake of IMI, TAL and VEL in Mo from 4 untreated GD patients. However, the intra-monocyte (IMo) GCA of TAL was systematically lower than that of IMI and VEL.Case analysis showed an inter-patient heterogeneity, with the highest increase of intra-monocyte enzyme activity for all REs in patient #2 and the lowest in patient #3; this observation was confirmed in vivo by analysis of Mo 15 min. after the end of the first IMI infusion (× 38 and × 9 endogeneous IMoGCA respectively) whereas patient #2 had received a lower dose of enzyme (45 U/kg/2 weeks vs 60 U/kg/2 weeks). Interestingly, patient #2 showed mild GD suffering only thrombocytopenia and asthenia while patient #3 had an aggressive form of GD, with bone disorders (aseptic osteonecrosis, bone infarction and pseudarthrosis after traumatic fracture). Patient #2 responded rapidly, with improved thrombocytopenia (68%) and increased hemoglobin level (+1.9g/dL) from M3 but hematological response for patient #3 could not be evaluated because initial parameters had been normal in this patient who had been splenectomized 20 years ago. Because of the chitotriosidase deficiency of patient #2, we used plasma CCL18 as a biomarker; its kinetic of decrease was clearly more rapid than for patient #3. Similarly, correction of glycosylated-ferritin was better than in patient #2. The two other patients with IMoGCA values close to that of patient #2 had non-progressive disease not requiring treatment (patient #1), or had thrombocytopenia that improved over the expected period of time (patient #4). Discussion:In conclusion, this study shows inter-patient variability in the ability of blood Mo to store recombinant enzymes which to our knowledge has not been previously reported. This was confirmed in vivo 15 min. after the start of the infusion. This variability could partially explain the heterogeneity of GD response to enzyme replacement therapy. In this small series, the least aggressive disease corresponded to the highest intra-monocyte GCA and the most aggressive disease to the least intra-monocyte GCA. Six-month follow-up showed differences in change in biological biomarkers suggesting a relationship between intra-monocyte GCA and disease response. Furthermore in this in vitro GD Mo model, all the compounds available are not similar, even if their chemical structures are only slightly different with no relationship with the expression of the CD206 (data not shown). Another RE influx mechanism could exist and influence enzyme replacement therapy. These findings could help in customizing replacement therapy. Disclosures:Belmatoug:Genzyme: Consultancy; Shire: Research Funding. Berger:Shire: Consultancy; Genzyme: Consultancy, Research Funding.

Impact of Measures to Enhance the Value of Observational Surveys in Rare Diseases: The Fabry Outcome Survey (FOS)

BackgroundDisease registries are an important source of information on the natural history of rare diseases and the response to new therapies in a real-world setting. The value of the information, however, is directly related to the completeness of the data entered for each patient over the course of time. The Fabry Outcome Survey (FOS) is a Shire Human Genetic Therapies–sponsored, physician-directed registry of patients with Fabry disease, a rare, multisystem, lysosomal storage disorder, established in 2001. Objective and MethodsIn 2005, measures were introduced to improve the completeness of data capture, including a focus on centers with 20 or more patients enrolled in the FOS, concentration on a limited number of core variables (i.e., serum creatinine, urinary protein, left ventricular mass [echocardiography], blood pressure [systolic and diastolic], pain, quality of life, and other Fabry disease–related signs and symptoms, as well as height and weight) and the introduction of Clinical Project Associates (CPAs) to facilitate data management by participating treatment centers. ResultsAn analysis of random samples of approximately 25% of patients in the registry in 2008 showed significant increases in data capture for most of the core variables examined. ConclusionsWe conclude that the measures introduced in 2005 significantly improved the value of the information in the registry, which has contributed greatly to our understanding of patients' real-world experience with enzyme replacement therapy for Fabry disease.

Response of women with Fabry disease to enzyme replacement therapy: Comparison with men, using data from FOS—the Fabry Outcome Survey

Fabry disease (α-galactosidase A deficiency) is an X-linked disorder. Women who are heterozygous for disease-causing mutations often manifest signs and symptoms of Fabry disease, but most studies of the effects of enzyme replacement therapy (ERT) have included only men. To date, no direct comparison has been made of the relative effectiveness of long-term ERT between men and women. The aim of this analysis was to report the effectiveness of agalsidase alfa in a cohort of 78 women treated for 4 years and to compare outcomes with those of 172 men. All data were obtained from the Fabry Outcome Survey—an international database of patients with Fabry disease sponsored by Shire Human Genetic Therapies. Quantifiable clinical parameters were assessed at baseline and the 4-year time point. Measures of pain, health-related quality of life, cardiac structure and function, and renal function changed to a similar extent in women and men during treatment, with the exception of left ventricular mass, which only reduced significantly in women. Changes in the presence of each of 27 clinical features after 4 years of ERT were evaluated in two subpopulations: patients with and patients without clinical features at baseline. It was clear for most types of clinical features that a number of women with a feature at baseline were no longer reported to have it at the 4-year time point, and that clinical features were observed in only a small percentage of women in whom they had been absent at baseline. The percentage of patients who were symptomatic at the 4-year time point was calculated for each type of clinical feature. The results showed no significant differences between men and women for most clinical features evaluated. Overall, both sexes responded to agalsidase alfa in a similar way, suggesting there should be no difference in the criteria for assessment of treatment in women and men.

Modelling the resource implications of managing adults with Fabry disease in Italy

This study estimated the resource implications and budget impact of managing adults with Fabry disease in Italy, from the perspective of the Servizio Sanitario Nazionale (SSN). A decision model was constructed using published clinical outcomes and clinician-derived resource utilisation estimates depicting the management of adults with Fabry disease in Italy. The expected annual cost of managing 220 existing and 20 new Fabry patients in Italy was estimated to be €28·3 million. In an average year, patients receiving enzyme replacement therapy (ERT) with 0·2 mg kg(-1) agalsidase alfa (Replagal; Shire Human Genetic Therapies, Basingstoke, Hampshire, UK) or 1·0 mg kg(-1) agalsidase beta (Fabrazyme; Genzyme Europe BV, Naarden, The Netherlands) are collectively expected to make 4500 hospital attendances to a day ward for infusions, which equates to 2000 eight-h days on the day ward associated with ERT. If all ERT-treated patients received their infusions at home, there would be a marginal reduction in the annual health care cost to manage these patients, and the total annual number of days on the day ward associated with ERT in the second year could potentially be reduced from a mean 2000 to zero, thereby releasing substantial hospital resources for use by non-Fabry patients. Currently, only agalsidase alfa is licensed for home treatment in Italy; hence, only patients receiving this enzyme could be offered home treatment. Use of agalsidase alfa (0·2 mg kg(-1) ) instead of agalsidase beta (1·0 mg kg(-1)) has the potential to reduce health care costs and release hospital resources in different specialities for alternative use by non-Fabry patients, thereby improving the efficiency of the public health care system in Italy.

The importance of mentors in a 20‐year gestation

American Journal of Medical Genetics Part AVolume 152A, Issue 11 p. 2695-2696 Invited Comment The importance of mentors in a 20-year gestation† Rick A. Martin, Corresponding Author Rick A. Martin [email protected] Shire Human Genetic Therapies, Boston, MassachusettsShire Human Genetic Therapies, Boston, MA.Search for more papers by this author Rick A. Martin, Corresponding Author Rick A. Martin [email protected] Shire Human Genetic Therapies, Boston, MassachusettsShire Human Genetic Therapies, Boston, MA.Search for more papers by this author First published: 14 October 2010 https://doi.org/10.1002/ajmg.a.33728 † How to Cite this Article: Martin RA. 2010. The Importance of mentors in a 20-year gestation. Am J Med Genet Part A 152A:2695–2696. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. Volume152A, Issue11November 2010Pages 2695-2696 RelatedInformation

Scope and management of Patient Registries for orphan rare disease (ORD) meeting the demands of all involved stakeholders

Once a product for an ORD is approved; conditional or non conditional, there are still unanswered questions on long-term safety and effectiveness. And there is often limited knowledge on natural progression of the disease and optimal disease management. These questions could be answered by prospectively collecting real world data into patient registries. The scope of the registry should be in line with demands from registry stakeholders such as regulatory agencies, payers, patients, treating physicians and the pharmaceutical industry. Fabry and Hunter Outcome Surveys (FOS and HOS) supported by Shire Human Genetic Therapies have been designed and managed for almost 10 years in close collaboration with stakeholders. Patient registries do meet challenges related to data completeness and quality. Treating physicians are not mandated to provide real world data to patient registries. Over the years, global guidance on good practices for design, conduct, analysis and reporting of patient registry data have been developed to increase the quality and facilitate the usage of patient registry data. In addition, specific initiatives from FOS and HOS Governance bodies have been undertaken to increase the robustness of the collected data. FOS and HOS have contributed to increased disease knowledge and improved disease management. Examples on findings are increased knowledge of the natural progression of the diseases, disease involvement in patient populations not eligible for inclusion into the initial clinical trials as well as long term clinical benefit of treatment. However there are still questions to be addressed and answered especially to support pricing and reimbursement decisions in some European countries. This is to insure patients gets access to equal treatment on approved treatment independent of where they live in Europe. Questions that need to be more closely addressed and evaluated are beyond clinical effectiveness and related to patients capacity to work and implications on public spending for these ORD.

First experience of enzyme replacement therapy with idursulfase in Spanish patients with Hunter syndrome under 5 years of age: Case observations from the Hunter Outcome Survey (HOS)

Hunter syndrome (mucopolysaccharidosis type II [MPS II], OMIM309900) is a rare X-linked lysosomal storage disorder caused by deficiency of the enzyme iduronate-2-sulphatase, resulting in accumulation of glycosaminoglycans (GAGs), multisystem organ failure and early death. Enzyme replacement therapy (ERT) with idursulfase is commercially available since 2007. Early access programs were established since 2005. However, limited information on the effects of ERT in young children is available to date. The aim of this analysis was therefore to determine the effects of ERT on patients younger than 5 years of age. We report data from six Spanish patients with confirmed Hunter syndrome who were younger than 5 years at the start of ERT, and had been treated with weekly intravenous infusions of idursulfase between 6 and 14 months. Baseline and treatment data were obtained from the Hunter Outcome Survey (HOS). HOS is an international database of MPS II patients on ERT or candidates to be treated, that collects data in a registry manner. HOS is supported by Shire Human Genetic Therapies, Inc. (Cambridge, MA, USA). At baseline, all patients showed neurological abnormalities, including ventriculomegaly, hydrocephaly, cerebral atrophy, perivascular changes and white matter lesions. Other signs and symptoms included thoracic deformity, otitis media, joint stiffness and hepatosplenomegaly, demonstrating that children under 5 years old can also be severely affected. ERT reduced urinary GAG levels, and reduced spleen ( n = 2) and liver size ( n = 1) after only 8 months. Height growth was maintained within the normal range during ERT. Joint mobility either stabilized or improved during ERT. In conclusion, this case series confirms the early onset of signs and symptoms of Hunter syndrome and provides the first evidence of ERT beneficial effects in patients less than 5 years of age. Similar efficacy and safety profiles to those seen in older children can be suggested, although further studies including a direct comparison with older patients would still be required.

Successful treatment of acquired C1 inhibitor deficiency with icatibant

C1 inhibitor is a major regulator of kallikrein and of C1r and C1s in the early classical complement pathway. Deficiency of C1 inhibitor results in overactivation of kallikrein, causing excess bradykinin production and localized cutaneous or mucosal oedema. Icatibant (Firazyr; Shire Human Genetic Therapies, Basingstoke, Hampshire, UK) was licensed in Europe in July 20081 for the treatment of acute attacks of hereditary angio‐oedema (C1 inhibitor deficiency). It is an antagonist of bradykinin B2 receptors and blocks the effects of endogenous bradykinin. It is given subcutaneously and may have a role in home treatment, although it is currently only licensed to be administered by a healthcare professional. We present a case of acquired C1 inhibitor deficiency (acquired angio‐oedema; AAE) that was successfully treated with icatibant. The patient was a 49‐year‐old man with acquired C1 inhibitor deficiency related to an IgG λ (1 g/L) monoclonal gammopathy of uncertain significance, not requiring specific treatment. He has attacks of angio‐oedema approximately once per month, which if untreated last 3–5 days. Mild attacks respond to tranexamic acid and more severe attacks respond fully to C1 inhibitor infusion 1000 U, with response starting within 30–120 min.