Recombinant Tissue-nonspecific Alkaline Phosphatase Research Articles

Prenatal asfotase alfa-mediated enzyme replacement therapy restores delayed calcification in a severe infantile form of hypophosphatasia model mice

Hypophosphatasia (HPP) is a congenital disorder caused by mutations in the tissue-nonspecific alkaline phosphatase (TNALP) gene. The pathogenesis of HPP varies, ranging from severe cases in which there is total absence of fetal bone calcification, which leads to stillbirth, to relatively mild cases in which the effects are confined to the teeth, such as early loss of the primary teeth. In recent years, the establishment of enzyme supplementation as a treatment method has prolonged survival in patients; however, this approach does not provide sufficient improvement for failed calcification. Furthermore, the effects of enzyme replacement therapy on the jawbone and periodontal tissues have not yet been studied in detail. Therefore, in this study, we investigated the therapeutic effects of enzyme replacement therapy on jawbone hypocalcification in mice. Recombinant TNALP was administered to mothers before birth and newborns immediately after birth, and the effect of treatment was evaluated at 20 days of age. The treated HPP mice had improved mandible (mandibular length and bone quality) and tooth quality (root length of mandibular first molar, formation of cementum), as well as improved periodontal tissue structure (structure of periodontal ligament). Furthermore, prenatal treatment had an additional therapeutic effect on the degree of mandible and enamel calcification. These results suggest that enzyme replacement therapy is effective for the treatment of HPP, specifically in the maxillofacial region (including the teeth and mandible), and that early initiation of treatment may have additional beneficial therapeutic effects.

Imaging patterns in pediatric hypophosphatasia.

Hypophosphatasia is a rare genetic disorder of calcium and phosphate metabolism due to ALPL gene mutations, which leads to abnormal mineralization of the bones and teeth. Hypophosphatasia is characterized by low serum alkaline phosphatase activity and a number of clinical signs, including failure to thrive, bone pain and dental issues. The diagnosis is suspected based on clinical, laboratory and imaging findings and confirmed by genetic testing. Diagnosis in children is often delayed due to a lack of disease awareness, despite specific imaging findings that are a cornerstone of the diagnosis. The recent approval of enzyme replacement therapy (bone-targeted recombinant tissue nonspecific alkaline phosphatase) has given imaging an important role in monitoring treatment efficacy. The aim of this pictorial essay is to review the imaging features of hypophosphatasia at diagnosis and during follow-up, including whole-body magnetic resonance imaging patterns.

TNAP: A New Multitask Enzyme in Energy Metabolism.

Tissue-nonspecific alkaline phosphatase (TNAP) is mainly known for its necessary role in skeletal and dental mineralization, which relies on the hydrolysis of the mineralization inhibitor inorganic pyrophosphate (PPi). Mutations in the gene encoding TNAP leading to severe hypophosphatasia result in strongly reduced mineralization and perinatal death. Fortunately, the relatively recent development of a recombinant TNAP with a bone anchor has allowed to correct the bone defects and prolong the life of affected babies and children. Researches on TNAP must however not be slowed down, because accumulating evidence indicates that TNAP activation in individuals with metabolic syndrome (MetS) is associated with enhanced cardiovascular mortality, presumably in relation with cardiovascular calcification. On the other hand, TNAP appears to be necessary to prevent the development of steatohepatitis in mice, suggesting that TNAP plays protective roles. The aim of the present review is to highlight the known or suspected functions of TNAP in energy metabolism that may be associated with the development of MetS. The location of TNAP in liver and its function in bile excretion, lipopolysaccharide (LPS) detoxification and fatty acid transport will be presented. The expression and function of TNAP in adipocyte differentiation and thermogenesis will also be discussed. Given that TNAP is a tissue- and substrate-nonspecific phosphatase, we believe that it exerts several crucial pathophysiological functions that are just beginning to be discovered.

Prenatal enzyme replacement therapy for Akp2 -/- mice with lethal hypophosphatasia.

Hypophosphatasia (HPP) is a congenital skeletal disease. Impairment of bone mineralization and seizures are due to a deficiency of tissue-nonspecific alkaline phosphatase (TNAP). Enzyme replacement therapy (ERT) is available as a highly successful treatment for pediatric-onset HPP. However, the potential for prenatal ERT has not been fully investigated to date. In this study, we assessed outcomes and maternal safety using a combinational approach with prenatal and postnatal administration of recombinant TNAP in Akp2−/− mice as a model of infantile HPP. For the prenatal ERT, we administered subcutaneous injections of recombinant TNAP to pregnant mice from embryonic day 11.5–14.5 until delivery, and then sequentially to Akp2−/− pups from birth to day 18. For the postnatal ERT, we injected Akp2−/− pups from birth until day 18. Prenatal ERT did not cause any ectopic mineralization in heterozygous maternal mice. Both prenatal and postnatal ERT preserved growth, survival rate and improved bone calcification in Akp2−/− mice. However, the effects of additional prenatal treatment to newborn mice appeared to be minimal, and the difference between prenatal and postnatal ERT was subtle. Further improvement of the prenatal ERT schedule and long-term observation will be required. The present paper sets a standard for such future studies.

Gene Therapy Using Adeno-Associated Virus Serotype 8 Encoding TNAP-D10 Improves the Skeletal and Dentoalveolar Phenotypes in Alpl-/- Mice.

ABSTRACTHypophosphatasia (HPP) is caused by loss‐of‐function mutations in the ALPL gene that encodes tissue‐nonspecific alkaline phosphatase (TNAP), whose deficiency results in the accumulation of extracellular inorganic pyrophosphate (PPi), a potent mineralization inhibitor. Skeletal and dental hypomineralization characterizes HPP, with disease severity varying from life‐threatening perinatal or infantile forms to milder forms that manifest in adulthood or only affect the dentition. Enzyme replacement therapy (ERT) using mineral‐targeted recombinant TNAP (Strensiq/asfotase alfa) markedly improves the life span, skeletal phenotype, motor function, and quality of life of patients with HPP, though limitations of ERT include frequent injections due to a short elimination half‐life of 2.28 days and injection site reactions. We tested the efficacy of a single intramuscular administration of adeno‐associated virus 8 (AAV8) encoding TNAP‐D10 to increase the life span and improve the skeletal and dentoalveolar phenotypes in TNAP knockout (Alpl −/−) mice, a murine model for severe infantile HPP. Alpl −/− mice received 3 × 1011 vector genomes/body of AAV8‐TNAP‐D10 within 5 days postnatal (dpn). AAV8‐TNAP‐D10 elevated serum ALP activity and suppressed plasma PPi. Treatment extended life span of Alpl −/− mice, and no ectopic calcifications were observed in the kidneys, aorta, coronary arteries, or brain in the 70 dpn observational window. Treated Alpl −/− mice did not show signs of rickets, including bowing of long bones, enlargement of epiphyses, or fractures. Bone microstructure of treated Alpl −/− mice was similar to wild type, with a few persistent small cortical and trabecular defects. Histology showed no measurable osteoid accumulation but reduced bone volume fraction in treated Alpl −/− mice versus controls. Treated Alpl −/− mice featured normal molar and incisor dentoalveolar tissues, with the exceptions of slightly reduced molar enamel and alveolar bone density. Histology showed the presence of cementum and normal periodontal ligament attachment. These results support gene therapy as a promising alternative to ERT for the treatment of HPP. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Synthesis and computational studies of highly selective inhibitors of human recombinant tissue non-specific alkaline phosphatase (h-TNAP): A therapeutic target against vascular calcification

In this study, we have discovered small druglike molecules as selective inhibitors of human tissue-nonspecific alkaline phosphatase (h-TNAP), an enzyme critical for the regulation of extracellular matrix calcification. The upregulation of h-TNAP is associated with various pathologies particularly the vascular calcification (VC). Selective inhibition of h-TNAP over h-NPP1 may serve as a useful therapeutic strategy against vascular calcification. A series of novel triazolyl pyrazole derivatives (10a-y) in which thiol bearing triazole moiety as the zinc binding functional group was introduced to a pyrazole based pharmacophore was synthesized and evaluated as potent and selective inhibitors of h-TNAP over h-NPP1. The biological screening against h-TNAP, h-IAP, h-NPP1 and h-NPP3 showed that many of the synthesized compounds are selective inhibitors of TNAP. Particularly, the compounds 10a-h, 10j, 10m-q, 10u, 10w and 10x displayed high potency and complete selectivity towards h-TNAP over h-NPP1. Compound 10q emerged as a highly potent inhibitor (IC50 = 0.16 µM or 160 nM) against h-TNAP with 127-fold increased inhibition compared to levamisole. On the other hand, compound 10e was found to be most selective inhibitor against the tested APs and NPPs (IC50 = 1.59 ± 0.36 µM). Binding sites architecture analysis, molecular-docking and molecular dynamics simulations (MDS), revealed the basis for h-TNAP and h-IAP ligand selectivity as well as selectivity towards h-TNAP over h-NPP1. These newly discovered inhibitors are believed to represent valuable lead structures to further streamline the generation of candidate compounds to target VC.

Viral delivery of tissue nonspecific alkaline phosphatase diminishes craniosynostosis in one of two FGFR2C342Y/+ mouse models of Crouzon syndrome.

Craniosynostosis is the premature fusion of cranial bones. The goal of this study was to determine if delivery of recombinant tissue nonspecific alkaline phosphatase (TNAP) could prevent or diminish the severity of craniosynostosis in a C57BL/6 FGFR2C342Y/+ model of neonatal onset craniosynostosis or a BALB/c FGFR2C342Y/+ model of postnatal onset craniosynostosis. Mice were injected with a lentivirus encoding a mineral targeted form of TNAP immediately after birth. Cranial bone fusion as well as cranial bone volume, mineral content and density were assessed by micro CT. Craniofacial shape was measured with calipers. Alkaline phosphatase, alanine amino transferase (ALT) and aspartate amino transferase (AST) activity levels were measured in serum. Neonatal delivery of TNAP diminished craniosynostosis severity from 94% suture obliteration in vehicle treated mice to 67% suture obliteration in treated mice, p<0.02) and the incidence of malocclusion from 82.4% to 34.7% (p<0.03), with no effect on cranial bone in C57BL/6 FGFR2C342Y/+ mice. In contrast, treatment with TNAP increased cranial bone volume (p< 0.01), density (p< 0.01) and mineral content (p< 0.01) as compared to vehicle treated controls, but had no effect on craniosynostosis or malocclusion in BALB/c FGFR2C342Y/+ mice. These results indicate that postnatal recombinant TNAP enzyme therapy diminishes craniosynostosis severity in the C57BL/6 FGFR2C342Y/+ neonatal onset mouse model of Crouzon syndrome, and that effects of exogenous TNAP are genetic background dependent.

MON-497 Adult Onset Hypophosphatasia: Before and After Treatment with Asfotase Alfa

Hypophosphatasia is a rare inherited bone disease resulting from mutations in the gene encoding tissue-nonspecific alkaline phosphatase (TNSALP), an enzyme predominant in skeleton, liver, kidney and teeth. Diminished TNSALP activity causes accumulation of substrates that inhibit bone mineralization resulting in debilitating pain, fractures and low alkaline phosphatase (ALP) levels. In 2015 the FDA approved asfotase alfa, a bone-targeted recombinant TNSALP used for enzyme replacement therapy. We present a case of hypophosphatasia treated with asfotase alfa resulting in improvements in whole body scan and pedometer step counts. In 2014, a 52-year-old female with chronic pain presented with over 30 years of bony pain, poor balance, falls, fractures and life-long dental disease. Physical exam displayed tenderness of long bones and waddling gait. Blood work was remarkable for low ALP at 11 U/L (normal 35-120 U/L) and low bone-specific ALP of 3 ug/L (normal 7.0-22.4 ug/L). Vitamin B6 level was >2000 nmol/L (normal 20.0-125.0 nmol/L). Calcium level was 9.8 mg/dL (normal 8.5-10.1 mg/dL). Serum phosphorus was elevated at 5.2 mg/dL (normal 2.3-4.6 mg/dL). Intact parathyroid hormone level was elevated at 114.1 pg/mL (normal 18.5-88.0 pg/mL), with known diagnosis of chronic kidney disease, GFR 27 mL/min. Her 24-hour urine calcium, serum protein electrophoresis, celiac test, thyroid studies and vitamin D level were normal. N-telopeptide was elevated at 25.9 nM (normal 6.2 to 19.0 nM). SPECT whole body scan demonstrated increased uptake with multiple fractures of the axial skeleton and proximal femurs; consistent with oncogenic osteomalacia. Fibroblast growth factor 23 and indium 111 octreotide scan showed no evidence of malignancy. DEXA scan showed osteopenia of the hip and forearm. She was diagnosed with adult-onset hypophosphatasia. Initially, due to lack of options, she was treated with denosumab. After three injections, she developed a new atypical femur fracture requiring surgical rod placement; denosumab was discontinued. In 2017, she was started on asfotase alfa with significant improvements in balance, endurance, and bone pain. Prior to treatment, the patient required narcotic oral pain medicine, fentanyl patch and an assistive device to walk 3000 steps daily; after treatment, she is walking over 10,000 steps daily without any pain pharmacotherapy. Repeat whole body scan showed less focal uptake overall, consistent with healing fractures. This case highlights clinical proof of improvement after treatment with asfotase alfa based on increased daily step count and healing fractures on whole body scans. Asfotase alfa is a newly approved therapy for hypophosphatasia and therefore long-term outcomes are not yet available, however short term patient reported subjective findings and clinical data are reassuring for successful treatment.

Tissue Nonspecific Alkaline Phosphatase (TNAP) Regulates Cranial Base Growth and Synchondrosis Maturation

Hypophosphatasia is a rare heritable disorder caused by inactivating mutations in the gene (Alpl) that encodes tissue nonspecific alkaline phosphatase (TNAP). Hypophosphatasia with onset in infants and children can manifest as rickets. How TNAP deficiency leads to bone hypomineralization is well explained by TNAP's primary function of pyrophosphate hydrolysis when expressed in differentiated bone forming cells. How TNAP deficiency leads to abnormalities within endochondral growth plates is not yet known. Previous studies in hypophosphatemic mice showed that phosphate promotes chondrocyte maturation and apoptosis via MAPK signaling. Alpl−/− mice are not hypophosphatemic but TNAP activity does increase local levels of inorganic phosphate. Therefore, we hypothesize that TNAP influences endochondral bone development via MAPK. In support of this premise, here we demonstrate cranial base bone growth deficiency in Alpl−/− mice, utilize primary rib chondrocytes to show that TNAP influences chondrocyte maturation, apoptosis, and MAPK signaling in a cell autonomous manner; and demonstrate that similar chondrocyte signaling and apoptosis abnormalities are present in the cranial base synchondroses of Alpl−/− mice. Micro CT studies revealed diminished anterior cranial base bone and total cranial base lengths in Alpl−/− mice, that were prevented upon injection with mineral-targeted recombinant TNAP (strensiq). Histomorphometry of the inter-sphenoidal synchondrosis (cranial base growth plate) demonstrated significant expansion of the hypertrophic chondrocyte zone in Alpl−/− mice that was minimized upon treatment with recombinant TNAP. Alpl−/− primary rib chondrocytes exhibited diminished chondrocyte proliferation, aberrant mRNA expression, diminished hypertrophic chondrocyte apoptosis and diminished MAPK signaling. Diminished apoptosis and VEGF expression were also seen in 15 day-old cranial base synchondroses of Alpl−/− mice. MAPK signaling was significantly diminished in 5 day-old cranial base synchondroses of Alpl−/− mice. Together, our data suggests that TNAP is essential for the later stages of endochondral bone development including hypertrophic chondrocyte apoptosis and VEGF mediated recruitment of blood vessels for replacement of cartilage with bone. These changes may be mediated by diminished MAPK signaling in TNAP deficient chondrocytes due to diminished local inorganic phosphate production.

Asfotase Alfa in Perinatal/Infantile-Onset and Juvenile-Onset Hypophosphatasia: A Guide to Its Use in the USA.

Subcutaneous asfotase alfa (Strensiq™), a first-in-class bone-targeted human recombinant tissue-nonspecific alkaline phosphatase (TNSALP) replacement therapy, is approved in the USA for the treatment of patients with perinatal/infantile- or juvenile-onset hypophosphatasia (HPP). In clinical trials, asfotase alfa was an effective and generally well tolerated treatment for perinatal/infantile- and juvenile onset-HPP through at least 3 and 5 years' treatment, respectively. Relative to untreated age-matched, juvenile-onset-HPP historical control cohorts, survival and ventilation-free survival were significantly prolonged in asfotase alfa-treated patients, consequent to preceding improvements in bone mineralization.

Asfotase Alfa: A Review in Paediatric-Onset Hypophosphatasia.

Hypophosphatasia (HPP) is a rare inheritable disease that results from loss-of-function mutations in the ALPL gene encoding tissue-nonspecific alkaline phosphatase (TNSALP). Therapeutic options for treating the underlying pathophysiology of the disease have been lacking, with the mainstay of treatment being management of symptoms and supportive care. HPP is associated with significant morbidity and mortality in paediatric patients, with mortality rates as high as 100% in perinatal-onset HPP and 50% in infantile-onset HPP. Subcutaneous asfotase alfa (Strensiq(®)), a first-in-class bone-targeted human recombinant TNSALP replacement therapy, is approved in the EU for long-term therapy in patients with paediatric-onset HPP to treat bone manifestations of the disease. In noncomparative clinical trials in infants and children with paediatric-onset HPP, asfotase alfa rapidly improved radiographically-assessed rickets severity scores at 24weeks (primary timepoint) as reflected in improvements in bone mineralization, with these benefits sustained after more than 3years of treatment. Furthermore, patients typically experienced improvements in respiratory function, gross motor function, fine motor function, cognitive development, muscle strength (normalization) and ability to perform activities of daily living, and catch-up height-gain. In life-threatening perinatal and infantile HPP, asfotase alfa also improved overall survival. Asfotase alfa was generally well tolerated in clinical trials, with relatively few patients discontinuing treatment and most treatment-related adverse events being of mild to moderate intensity. Thus, subcutaneous asfotase alfa is a valuable emerging therapy for the treatment of bone manifestations in patients with paediatric-onset HPP.

The effects of tissue-non-specific alkaline phosphatase gene therapy on craniosynostosis and craniofacial morphology in the FGFR2C342Y/+ mouse model of Crouzon craniosynostosis.

Craniosynostosis, the premature fusion of cranial bones, has traditionally been described as a disease of increased bone mineralization. However, multiple mouse models of craniosynostosis display craniosynostosis simultaneously with diminished cranial bone volume and/or density. We propose an alternative hypothesis that craniosynostosis results from abnormal tissue mineralization through the downregulation of tissue-non-specific alkaline phosphatase (TNAP) enzyme downstream of activating mutations in FGFRs. Neonatal Crouzon (FGFRC342Y/+) and wild-type (FGFR+/+) mice were injected with lentivirus to deliver a recombinant form of TNAP. Mice were sacrificed at 4 weeks postnatal. Serum was collected to test for alkaline phosphatase (AP), phosphorus, and calcium levels. Craniofacial bone fusion and morphology were assessed by micro-computed tomography. Injection with the TNAP lentivirus significantly increased serum AP levels (increased serum AP levels are indicative of efficient transduction and production of the recombinant protein), but results were variable and dependent upon viral lot and the litter of mice injected. Morphological analysis revealed craniofacial form differences for inferior surface (p=0.023) and cranial height (p=0.014) regions between TNAP lentivirus-injected and vehicle-injected Crouzon mice. With each unit increase in AP level, the odds of lambdoid suture fusion decreased by 84.2% and these results came close to statistical significance (p=0.068). These results suggest that TNAP deficiency may mediate FGFR2-associated craniosynostosis. Future studies should incorporate injection of recombinant TNAP protein, to avoid potential side effects and variable efficacy of lentiviral gene delivery.

Improvement of the skeletal and dental hypophosphatasia phenotype in Alpl−/− mice by administration of soluble (non-targeted) chimeric alkaline phosphatase

Hypophosphatasia (HPP) results from ALPL gene mutations, which lead to a deficiency of tissue-nonspecific alkaline phosphatase (TNAP), and accumulation of inorganic pyrophosphate, a potent inhibitor of mineralization that is also a natural substrate of TNAP, in the extracellular space. HPP causes mineralization disorders including soft bones (rickets or osteomalacia) and defects in teeth and periodontal tissues. Enzyme replacement therapy using mineral-targeting recombinant TNAP has proven effective in preventing skeletal and dental defects in TNAP knockout (Alpl−/−) mice, a model for life-threatening HPP. Here, we show that the administration of a soluble, intestinal-like chimeric alkaline phosphatase (ChimAP) improves the manifestations of HPP in Alpl−/− mice. Mice received daily subcutaneous injections of ChimAP at doses of 1, 8 or 16mg/kg, from birth for up to 53days. Lifespan and body weight of Alpl−/− mice were normalized, and vitamin B6-associated seizures were absent with 16mg/kg/day of ChimAP. Radiographs, μCT and histological analyses documented improved mineralization in cortical and trabecular bone and secondary ossification centers in long bones of ChimAP16-treated mice. There was no evidence of craniosynostosis in the ChimAP16-treated mice and we did not detect ectopic calcification by radiography and histology in the aortas, stomachs, kidneys or lungs in any of the treatment groups. Molar tooth development and function improved with the highest ChimAP dose, including enamel, dentin, and tooth morphology. Cementum remained deficient and alveolar bone mineralization was reduced compared to controls, though ChimAP-treated Alpl−/− mice featured periodontal attachment and retained teeth. This study provides the first evidence for the pharmacological efficacy of ChimAP for use in the treatment of skeletal and dental manifestations of HPP.

Bone Marrow Cell Based Enzyme Replacement Prolongs Survival and Improves Disease Phenotypes In a Mouse Model Of Lethal Hypophosphatasia

Hypophosphatasia (HPP) is an inherited skeletal disease caused by mutations of the ALPL gene that encodes tissue-nonspecific alkaline phosphatase (TNALP). TNALP is an ectoenzyme and plays an essential role in bone mineralization. The major symptoms of HPP are hypomineralization of systemic bones, respiratory insufficiency and epileptic seizures. Perinatal and infantile forms of HPP are often fatal. Since ALP functions on the exterior of the cells, enzyme replacement therapy (ERT) is a potential approach to treat HPP. Currently, Phase II/III clinical trials of ERT using a recombinant TNALP which linked deca-aspartate (D10) at the C terminus for bone targeting are ongoing in North America, Europe and Japan. The perinatal and infantile patients received the ERT showed apparent improvement of the symptoms. However, the ERT is highly invasive for the young patients because it requires repeated subcutaneous administration of large amounts of the enzyme every 3 times a week for long-term correction.As another approach to treat HPP, we have reported in vivo gene therapy for ALPL (Akp2) knock-out mice (HPP mice). The treated HPP mice were rescued by a single systemic injection of lentiviral vector or adeno-associated viral vector expressing bone targeted form of TNALP (TNALP-D10) during the neonatal or fetal period. Although untreated HPP mice developed apparent growth failure and died by around 20 days of age due to severe skeletal hypomineralization and epileptic seizure, the treated HPP mice were prolonged the survival and improved the physical activity. In the treated HPP mice, plasma ALP activity was kept higher than 1 U/ml (approximately 0.01 U/ml in untreated HPP mice and 0.1 U/ml in wild type (WT) mice) which gives therapeutic effects. However, disadvantages of in vivo gene therapy include the risk of germline gene transfer and induction of immune responses to the vectors or transgene products.To overcome these problems, we examined a feasibility of ex vivo gene therapy using hematopoietic stem cells (HSC) transduced by lentiviral vector expressing TNALP-D10. The potential advantages of this approach are lifelong expression of TNALP-D10 and prevention of risks of in vivo gene therapy. The lineage negative bone marrow cells containing HSC (Lin- BMC) were harvested from B6.CD45.1 mice (Ly5.1) and then enriched using Mouse Hematopoietic Progenitor (Stem) Cell Enrichment Set (BD bioscience). Lin- BMC was transduced with lentiviral vector expressing TNALP-D10 for 20 hrs at an moi of 50 with mSCF, mIL3 and rhIL6 on Retronectin coated plate. Recipient HPP mice (Ly5.2) on day 2 after birth were received a sub-lethal dose of total body irradiation (4Gy) 4hr prior to transplantation. Then, the transduced Lin- BMC (1 x 106 cells) was transplanted intravenously into the HPP mice through the temporal vein or jugular vein. The plasma ALP activity was rapidly elevated approximately 400 fold higher than untreated HPP mice (untreated: 0.014±0.004 units/ml (n=4) and treated: 5.39±2.29 units/ml (n=7), respectively) on 1 week after the transplantation and kept at this level during the observation period. Engraftment rate of Ly5.1 donor cells were sustained at approximately 30-40% with multilineage potential. The treated HPP mice were prolonged their survival over 3 months without epileptic seizures and the physical activities were improved. The histochemical ALP staining indicated TNALP-D10 was accumulated on the surface of trabecular and cortical bones of the treated HPP mice. The bone mineralization was significantly improved, but still not satisfactory compared with age matched WT mice. Contrary to our expectations, 2 of 9 HPP mice transplanted with non-transduced BMC also survived for 3 months. However, the plasma ALP activity was not elevated at all and the bone mineralization was incomplete compared with treated HPP mice.These results indicate that a single transplantation of genetically modified BMC at neonatal period is sufficient for long-term supply of TNALP-D10 and rescue of lethal HPP mice, even though hypomineralization was not completely recovered. Further optimization of viral vector and conditioning of transplantation is required to increase the treatment efficacy for HPP. However, neonatal ex vivo gene therapy using genetically modified BMC would be a possible and practical approach to treat HPP. Disclosures:Watanabe:Alexion Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees.

Enzyme replacement therapy on hypophosphatasia mouse model

Hypophosphatasia (HPP) is an inborn error of metabolism caused by deficiency of the tissue-nonspecific alkaline phosphatase (TNSALP), resulting in a defect of bone mineralization. Natural substrates for this ectoenzyme accumulate extracellulary including inorganic pyrophosphate (PPi), an inhibitor of mineralization, and pyridoxal 5-phosphate (PLP), a co-factor form of vitamin B6. Enzyme replacement therapy (ERT) for HPP by functional TNSALP is one of the therapeutic options. The C-terminal-anchorless human recombinant TNSALP derived from Chinese hamster ovary cell lines was purified. TNSALP-null mice (Akp2 (-/-) ), an infantile model of HPP, were treated from birth using TNSALP and vitamin B6 diet. Long-term efficacy studies of ERT consisted of every 3days subcutaneous or intravenous injections till 28days old (dose 20 U/g) and subsequently every 3days intravenous injections for 6months (dose 10 U/g). We assessed therapeutic effect by growth and survival rates, fertility, skeletal manifestations, and radiographic and pathological finding. Treated Akp2 (-/-) mice grew normally till 4weeks and appeared well with a minimum skeletal abnormality as well as absence of epilepsy, compared with untreated mice which died by 3weeks old. The prognosis of TNSALP-treated Akp2 (-/-) mice was improved substantially: 1) prolonged life span over 6months, 2) improvement of the growth, and 3) normal fertility. After 6months of treatment, we found moderate hypomineralization with abnormal proliferative chondrocytes in growth plate and articular cartilage. In conclusion, ERT with human native TNSALP improves substantial clinical manifestations in Akp2 (-/-) mice, suggesting that ERT with anchorless TNSALP is also a potential therapy for HPP.

Respiratory mechanics in an infant with perinatal lethal hypophosphatasia treated with human recombinant enzyme replacement therapy

Hypophosphatasia is a rare autosomal recessive disorder caused by deficient activity of tissue nonspecific alkaline phosphatase (TNSALP) and characterized by defective bone mineralization. In the perinatal lethal form, respiratory complications due to rachitic deformities of the thoracic cage and associated hypoplastic lungs are present. ENB-0040 is a bone-targeted human recombinant TNSALP fusion protein that aims to restore skeletal mineralization. The goal of this study was to characterize pulmonary and thoracic cage mechanics in an infant with the perinatal lethal form of hypophosphatasia under enzyme replacement therapy. Pulmonary function testing was performed on a preterm, 8-week-old patient with hypophosphatasia who was mechanically ventilated since birth because of severe chest wall insufficiency. The measurements consisted of respiratory impulse oscillation measurements (resistance and reactance), ventilatory mechanics (compliance and resistance), and thoracoabdominal motion (TAM) analysis. At baseline, chest wall compliance was 50% of normal, and the TAM indicated predominantly abdominal displacement. After 12 weeks of treatment, a consistent decrease in ventilator requirements and improvement in lung function and chest wall mechanics were observed and correlated with thoracic cage radiologic findings. Measurable changes in chest wall dynamics and respiratory mechanics using noninvasive technology were useful for respiratory management and therapeutic guidance of ENB-0040 treatment in this patient.

Dose response of bone-targeted enzyme replacement for murine hypophosphatasia

Hypophosphatasia (HPP) features rickets or osteomalacia from tissue-nonspecific alkaline phosphatase (TNSALP) deficiency due to deactivating mutations within the ALPL gene. Enzyme replacement therapy with a bone-targeted, recombinant TNSALP (sALP-FcD(10), renamed ENB-0040) prevents manifestations of HPP when initiated at birth in TNSALP knockout (Akp2(-/-)) mice. Here, we evaluated the dose-response relationship of ENB-0040 to various phenotypic traits of Akp2(-/-) mice receiving daily subcutaneous (SC) injections of ENB-0040 from birth at 0.5, 2.0, or 8.2mg/kg for 43days. Radiographs, μCT, and histomorphometric analyses documented better bone mineralization with increasing doses of ENB-0040. We found a clear, positive correlation between ENB-0040 dose and prevention of mineralization defects of the feet, rib cage, lower limbs, and jaw bones. According to a dose-response model, the ED(80) (the dose that prevents bone defects in 80% of mice) was 3.2, 2.8 and 2.9mg/kg/day for these sites, respectively. Long bones seemed to respond to lower daily doses of ENB-0040. There was also a positive relationship between ENB-0040 dose and survival. Median survival, body weight, and bone length all improved with increasing doses of ENB-0040. Urinary PP(i) concentrations remained elevated in all treatment groups, indicating that while this parameter is a good biochemical marker for diagnosing HPP in patients, it may not be a good follow up marker for evaluating response to treatment when administering bone-targeted TNSALP to mice. These dose-response relationships strongly support the pharmacological efficacy of ENB-0040 for HPP, and provide the experimental basis for the therapeutic range of ENB-0040 chosen for clinical trials.

Proteoliposomes Harboring Alkaline Phosphatase and Nucleotide Pyrophosphatase as Matrix Vesicle Biomimetics

We have established a proteoliposome system as an osteoblast-derived matrix vesicle (MV) biomimetic to facilitate the study of the interplay of tissue-nonspecific alkaline phosphatase (TNAP) and NPP1 (nucleotide pyrophosphatase/phosphodiesterase-1) during catalysis of biomineralization substrates. First, we studied the incorporation of TNAP into liposomes of various lipid compositions (i.e. in pure dipalmitoyl phosphatidylcholine (DPPC), DPPC/dipalmitoyl phosphatidylserine (9:1 and 8:2), and DPPC/dioctadecyl-dimethylammonium bromide (9:1 and 8:2) mixtures. TNAP reconstitution proved virtually complete in DPPC liposomes. Next, proteoliposomes containing either recombinant TNAP, recombinant NPP1, or both together were reconstituted in DPPC, and the hydrolysis of ATP, ADP, AMP, pyridoxal-5′-phosphate (PLP), p-nitrophenyl phosphate, p-nitrophenylthymidine 5′-monophosphate, and PPi by these proteoliposomes was studied at physiological pH. p-Nitrophenylthymidine 5′-monophosphate and PLP were exclusively hydrolyzed by NPP1-containing and TNAP-containing proteoliposomes, respectively. In contrast, ATP, ADP, AMP, PLP, p-nitrophenyl phosphate, and PPi were hydrolyzed by TNAP-, NPP1-, and TNAP plus NPP1-containing proteoliposomes. NPP1 plus TNAP additively hydrolyzed ATP, but TNAP appeared more active in AMP formation than NPP1. Hydrolysis of PPi by TNAP-, and TNAP plus NPP1-containing proteoliposomes occurred with catalytic efficiencies and mild cooperativity, effects comparable with those manifested by murine osteoblast-derived MVs. The reconstitution of TNAP and NPP1 into proteoliposome membranes generates a phospholipid microenvironment that allows the kinetic study of phosphosubstrate catabolism in a manner that recapitulates the native MV microenvironment.

ENB-0040, a bone-targeted human recombinant tissue nonspecific alkaline phosphatase (TNSALP) fusion protein, in the treatment of infants and adults with hypophosphatasia

ENB-0040, a bone-targeted human recombinant tissue nonspecific alkaline phosphatase (TNSALP) fusion protein, in the treatment of infants and adults with hypophosphatasia

Phenotype and Function of Alkaline Phosphatase Cells in a Murine Model of Radio-Induced Marrow Aplasia

Bone marrow aplasias are characterized by a peripheral blood cells deficit resulting from a deficiency in the production of one or several blood cells lineages in bone marrow. Some of these aplasias are able to regenerate, others don’t respond to treatment. In most of the cases, spontaneously regenerating bone marrows are characterized by the presence of a great number of fibroblastic cells presenting a strong membranous alkaline phosphatase activity (ALP), this activity has completely disappeared in irreversible bone marrow aplasias. The aim of our work is to analyze phenotypic and functional characteristics of these ALP positive reticular cells. In this aim, we used a murine radio-induced aplasia model: a 4 Gy gamma rays total body irradiation induced an important but reversible bone marrow aplasia. In a first step, we have demonstrated that the density of ALP network and the number of ALP positive cells increase strongly in the first three days following irradiation. This increase precede haematopoietic regeneration and isn’t a result of cell proliferation. In vitro studies have also demonstrated that this increase in ALP activity is due to the expression of TNSALP (tissue non specific alkaline phosphatase). Next, we have established a relationship between these ALP positive reticular cells and mesenchymal stem cells. In vitro observations, as well as phenotypic analysis, indicate that TNSALP+ cells differentiated from mesenchymal stem cells. Although expressing an early osteoblastic marker, cbfa1, they keep self-renewal and differentiation capacities. To test their function in haematopoiesis, we have realized co-cultures of CFU-Fs and haematopoietic precursors (ckit+, sca-1+, lin-). We observed that, CFU-Fs from irradiated bone marrow, characterized by a high proportion of TNSALP positive cells, strongly stimulate the proliferation and the differentiation to the myelo-monocytic and granulopoietic lineage. In accordance with this observation, bone marrows of TNSALP deficient mice, at a late embryonic stage, are characterized by a deficit in granulopoiesis. In order to understand the effects of TNSALP in granulopoiesis, we added recombinant TNSALP (Enobia, Montreal Canada) on TNSALP negative CFU-Fs from control mice and observed that it has an effect on the differentiation process without any effects on the proliferation. All these data suggest that TNSALP expressed by reticular cells, directly derived from mesenchymal stem cells plays a role in normal haematopoiesis and in regeneration of bone marrow aplasia by inducing differentiation of myelo-monocytic and granulocytic precursors. Recombinant TNSALP (Enobia, Montreal Canada) will be tested in murine models of bone marrow aplasias.