Novel treatment for PXE: Recombinant ENPP1 enzyme therapy.

Pseudoxanthoma elasticum (PXE) is a genetic multisystem ectopic calcification disorder caused by inactivating mutations in the ABCC6 gene encoding ABCC6, a hepatic efflux transporter. ABCC6-mediated ATP secretion by the liver is the main source of a potent endogenous calcification inhibitor, plasma inorganic pyrophosphate (PPi); the deficiency of plasma PPi underpins PXE. Recent studies demonstrated that INZ-701, a recombinant human ENPP1 that generates PPi and is now in clinical trials, restored plasma PPi levels and prevented ectopic calcification in the muzzle skin of Abcc6-/-mice. This study examined the pharmacokinetics, pharmacodynamics, and potency of a new ENPP1-Fc isoform, BL-1118, in Abcc6-/- mice. When Abcc6-/- mice received a single subcutaneous injection of BL-1118 at 0.25, 0.5, or 1 mg/kg, they had dose-dependent elevations in plasma ENPP1 enzyme activity and PPi levels, with an enzyme half-life of approximately 100 h. When Abcc6-/- mice were injected weekly from 5 to 15 weeks of age, BL-1118 dose-dependently increased steady-state plasma ENPP1 activity and PPi levels and significantly reduced ectopic calcification in the muzzle skin and kidneys. These results suggest that BL-1118 is a promising second generation enzyme therapy for PXE, the first generation of which is currently in clinical testing.

A dual-acting DNASE1/DNASE1L3 biologic prevents autoimmunity and death in genetic and induced lupus models.

A defining feature of systemic lupus erythematosus (SLE) is loss of tolerance to self-DNA, and deficiency of DNASE1L3, the main enzyme responsible for chromatin degradation in blood, is also associated with SLE. This association can be found in an ultrarare population of pediatric patients with DNASE1L3 deficiency who develop SLE, adult patients with loss-of-function variants of DNASE1L3 who are at a higher risk for SLE, and patients with sporadic SLE who have neutralizing autoantibodies against DNASE1L3. To mitigate the pathogenic effects of inherited and acquired DNASE1L3 deficiencies, we engineered a long-acting enzyme biologic with dual DNASE1/DNASE1L3 activity that is resistant to DNASE1 and DNASE1L3 inhibitors. Notably, we found that the biologic prevented the development of lupus in Dnase1-/-Dnase1L3-/- double-knockout mice and rescued animals from death in pristane-induced lupus. Finally, we confirmed that the human isoform of the enzyme biologic was not recognized by autoantibodies in SLE and efficiently degraded genomic and mitochondrial cell-free DNA, as well as microparticle DNA, in SLE plasma. Our findings suggest that autoimmune diseases characterized by aberrant DNA accumulation, such as SLE, can be effectively treated with a replacement DNASE tailored to bypass pathogenic mechanisms, both genetic and acquired, that restrict DNASE1L3 activity.

Quantitative correlation of ENPP1 pathogenic variants with disease phenotype.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) codes for a type 2 transmembrane glycoprotein which hydrolyzes extracellular phosphoanhydrides into bio-active molecules that regulate, inter alia, ectopic mineralization, bone formation, vascular endothelial proliferation, and the innate immune response. The clinical phenotypes produced by ENPP1 deficiency are disparate, ranging from life-threatening arterial calcifications to cutaneous hypopigmentation. To investigate associations between disease phenotype and enzyme activity we quantified the enzyme velocities of 29 unique ENPP1 pathogenic variants in 41 patients enrolled in an NIH study along with 33 other variants reported in literature. We correlated the relative enzyme velocities with the presenting clinical diagnoses, performing the catalytic velocity measurements simultaneously in triplicate using a high-throughput assay to reduce experimental variation. We found that ENPP1 variants associated with autosomal dominant phenotypes reduced enzyme velocities by 50 % or more, whereas variants associated with insulin resistance had non-significant effects on enzyme velocity. In Cole disease the catalytic velocities of ENPP1 variants associated with AD forms trended to lower values than those associated with autosomal recessive forms - 8-32 % vs. 33 % of WT, respectively. Additionally, ENPP1 variants leading to life-threatening vascular calcifications in GACI patients had widely variable enzyme activities, ranging from no significant differences compared to WT to the complete abolishment of enzyme velocity. Finally, disease severity in GACI did not correlate with the mean enzyme velocity of the variants present in affected compound heterozygotes but did correlate with the more severely damaging variant. In summary, correlation of ENPP1 enzyme velocity with disease phenotypes demonstrate that enzyme velocities below 50 % of WT levels are likely to occur in the context of autosomal dominant disease (due to a monoallelic variant), and that disease severity in GACI infants correlates with the more severely damaging ENPP1 variant in compound heterozygotes, not the mean velocity of the pathogenic variants present.

ENPP1 in Blood and Bone: Skeletal and Soft Tissue Diseases Induced by ENPP1 Deficiency.

The enzyme ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) codes for a type 2 transmembrane glycoprotein that hydrolyzes extracellular ATP to generate pyrophosphate (PPi) and adenosine monophosphate, thereby contributing to downstream purinergic signaling pathways. The clinical phenotypes induced by ENPP1 deficiency are seemingly contradictory and include early-onset osteoporosis in middle-aged adults and life-threatening vascular calcifications in the large arteries of infants with generalized arterial calcification of infancy. The progressive overmineralization of soft tissue and concurrent undermineralization of skeleton also occur in the general medical population, where it is referred to as paradoxical mineralization to highlight the confusing pathophysiology. This review summarizes the clinical presentation and pathophysiology of paradoxical mineralization unveiled by ENPP1 deficiency and the bench-to-bedside development of a novel ENPP1 biologics designed to treat mineralization disorders in the rare disease and general medical population.

Genetics of Diffuse Idiopathic Skeletal Hyperostosis and Ossification of the Spinal Ligaments.

PURPOSE OF REVIEW: The study aims to provide updated information on the genetic factors associated with the diagnoses 'Diffuse Idiopathic Skeletal Hyperostosis' (DISH), 'Ossification of the Posterior Longitudinal Ligament' (OPLL), and in patients with spinal ligament ossification. RECENT FINDINGS: Recent studies have advanced our knowledge of genetic factors associated with DISH, OPLL, and other spinal ossification (ossification of the anterior longitudinal ligament [OALL] and the yellow ligament [OYL]). Several case studies of individuals afflicted with monogenic disorders, such as X-linked hypophosphatemia (XLH), demonstrate the strong association of fibroblast growth factor 23-related hypophosphatemia with OPLL, suggesting that pathogenic variants in PHEX, ENPP1, and DMP1 are associated with FGF23-phosphate wasting phenotype and strong genetic factors placing patients at risk for OPLL. Moreover, emerging evidence demonstrates that heterozygous and compound heterozygous ENPP1 pathogenic variants inducing 'Autosomal Recessive Hypophosphatemic Rickets Type 2' (ARHR2) also place patients at risk for DISH and OPLL, possibly due to the loss of inhibitory plasma pyrophosphate (PPi) which suppresses ectopic calcification and enthesis mineralization. Our findings emphasize the importance of genetic and plasma biomarker screening in the clinical evaluation of DISH and OPLL patients, with plasma PPi constituting an important new biomarker for the identification of DISH and OPLL patients whose disease course may be responsive to ENPP1 enzyme therapy, now in clinical trials for rare calcification disorders.

Lonafarnib improves cardiovascular function and survival in a mouse model of Hutchinson-Gilford progeria syndrome.

Clinical trials have demonstrated that lonafarnib, a farnesyltransferase inhibitor, extends the lifespan in patients afflicted by Hutchinson-Gilford progeria syndrome, a devastating condition that accelerates many characteristics of aging and results in premature death due to cardiovascular sequelae. The US Food and Drug Administration approved Zokinvy (lonafarnib) in November 2020 for treating these patients, yet a detailed examination of drug-associated effects on cardiovascular structure, properties, and function has remained wanting. In this paper, we report encouraging outcomes of daily post-weaning treatment with lonafarnib on the composition and biomechanical phenotype of elastic and muscular arteries as well as associated cardiac function in a well-accepted mouse model of progeria that exhibits severe perimorbid cardiovascular disease. Lonafarnib resulted in 100% survival of the treated progeria mice to the study end-point (time of 50% survival of untreated mice), with associated improvements in arterial structure and function working together to significantly reduce pulse wave velocity and improve left ventricular diastolic function. By contrast, neither treatment with the mTOR inhibitor rapamycin alone nor dual treatment with lonafarnib plus rapamycin improved outcomes over that achieved with lonafarnib monotherapy.

Catalysis-Independent ENPP1 Protein Signaling Regulates Mammalian Bone Mass.

Biallelic ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) deficiency induces vascular/soft tissue calcifications in generalized arterial calcification of infancy (GACI), and low bone mass with phosphate-wasting rickets in GACI survivors (autosomal hypophosphatemic rickets type-2). ENPP1 haploinsufficiency induces early-onset osteoporosis and mild phosphate wasting in adults. Both conditions demonstrate the unusual combination of reduced accrual of skeletal mineral, yet excess and progressive heterotopic mineralization. ENPP1 is the only enzyme that generates extracellular pyrophosphate (PPi), a potent inhibitor of both bone and heterotopic mineralization. Life-threatening vascular calcification in ENPP1 deficiency is due to decreased plasma PPi; however, the mechanism by which osteopenia results is not apparent from an understanding of the enzyme's catalytic activity. To probe for catalysis-independent ENPP1 pathways regulating bone, we developed a murine model uncoupling ENPP1 protein signaling from ENPP1 catalysis, Enpp1T238A mice. In contrast to Enpp1asj mice, which lack ENPP1, Enpp1T238A mice have normal trabecular bone microarchitecture and favorable biomechanical properties. However, both models demonstrate low plasma Pi and PPi, increased fibroblast growth factor 23 (FGF23), and by 23 weeks, osteomalacia demonstrating equivalent phosphate wasting in both models. Reflecting findings in whole bone, calvarial cell cultures from Enpp1asj mice demonstrated markedly decreased calcification, elevated transcription of Sfrp1, and decreased nuclear ß-catenin signaling compared to wild-type (WT) and Enpp1T238A cultures. Finally, the decreased calcification and nuclear ß-catenin signaling observed in Enpp1asj cultures was restored to WT levels by knockout of Sfrp1. Collectively, our findings demonstrate that catalysis-independent ENPP1 signaling pathways regulate bone mass via the expression of soluble Wnt inhibitors such as secreted frizzled-related protein 1 (SFRP1), whereas catalysis dependent pathways regulate phosphate homeostasis through the regulation of plasma FGF23. © 2022 American Society for Bone and Mineral Research (ASBMR).

Characterization of hearing-impairment in Generalized Arterial Calcification of Infancy (GACI).

BACKGROUND AND IMPORTANCE: Hearing loss (HL) has been sporadically described, but not well characterized, in Generalized Arterial Calcification of Infancy (GACI), a rare disease in which pathological calcification typically presents in infancy. OBJECTIVES: This study aims to describe the clinical audiologic and otologic features and potential etiology of hearing impairment in GACI and gain pathophysiological insight from a murine model of GACI. DESIGN: Cross-sectional cohort study of individuals with GACI. Murine ossicle micromorphology of the ENPP1asj/asj mutant compared to wild-type. SETTING: Clinical research hospital; basic science laboratory. PARTICIPANTS: Nineteen individuals with GACI who met clinical, biochemical, and genetic criteria for diagnosis. MAIN OUTCOMES AND MEASURES: Clinical, biochemical, and radiologic features associated with hearing status. RESULTS: Pure-tone thresholds could be established in 15 (n = 30 ears) of the 19 patients who underwent audiological assessments. The prevalence of HL was 50% (15/30) of ears, with conductive HL in 80% and sensorineural HL in 20%. In terms of patients with HL (n = 8), seven patients had bilateral HL and one patient had unilateral HL. Degree of HL was mild to moderate for 87% of the 15 ears with hearing loss. Of those patients with sufficient pure-tone and middle ear function data, 80% (8/10) had audiometric configurations suggestive of ossicular chain dysfunction (OCD). Recurrent episodes of otitis media (ROM) requiring pressure-equalizing tube placement were common. In patients who underwent cranial CT, 54.5% (6/11) had auricular calcification. Quantitative backscattered electron imaging (qBEI) of murine ossicles supports an OCD component of auditory dysfunction in GACI, suggesting loss of ossicular osteocytes without initiation of bone remodeling. CONCLUSIONS AND RELEVANCE: Hearing loss is common in GACI; it is most often conductive, and mild to moderate in severity. The etiology of HL is likely multifactorial, involving dysfunction of the ossicular chain and/or recurrent otitis media. Clinically, this study highlights the importance of early audiologic and otologic evaluation in persons with GACI. Novel findings of high rates of OCD and ROM may inform management, and in cases of unclear HL etiology, dedicated temporal bone imaging should be considered.

Strategies for Glycoengineering Therapeutic Proteins.

Almost all therapeutic proteins are glycosylated, with the carbohydrate component playing a long-established, substantial role in the safety and pharmacokinetic properties of this dominant category of drugs. In the past few years and moving forward, glycosylation is increasingly being implicated in the pharmacodynamics and therapeutic efficacy of therapeutic proteins. This article provides illustrative examples of drugs that have already been improved through glycoengineering including cytokines exemplified by erythropoietin (EPO), enzymes (ectonucleotide pyrophosphatase 1, ENPP1), and IgG antibodies (e.g., afucosylated Gazyva®, Poteligeo®, Fasenra™, and Uplizna®). In the future, the deliberate modification of therapeutic protein glycosylation will become more prevalent as glycoengineering strategies, including sophisticated computer-aided tools for "building in" glycans sites, acceptance of a broad range of production systems with various glycosylation capabilities, and supplementation methods for introducing non-natural metabolites into glycosylation pathways further develop and become more accessible.

Identification of ENPP1 Haploinsufficiency in Patients With Diffuse Idiopathic Skeletal Hyperostosis and Early-Onset Osteoporosis.

Homozygous ENPP1 mutations are associated with autosomal recessive hypophosphatemic rickets type 2 (ARHR2), severe ossification of the spinal ligaments, and generalized arterial calcification of infancy type 1. There are a limited number of reports on phenotypes associated with heterozygous ENPP1 mutations. Here, we report a series of three probands and their families with heterozygous and compound heterozygous ENPP1 mutations. The first case (case 1) was a 47-year-old male, diagnosed with early-onset osteoporosis and low-normal serum phosphate levels, which invoked suspicion for hypophosphatemic rickets. The second and third cases were 77- and 54-year-old females who both presented with severe spinal ligament ossification and the presumptive diagnosis of diffuse idiopathic skeletal hyperostosis (DISH). Upon workup, fibroblast growth factor 23 (FGF23) was noted to be relatively high in case 2 and serum phosphorous was low-normal in case 3, and the diagnoses of X-linked hypophosphatemic rickets (XLH) and ARHR2 were considered. Genetic testing for genes related to congenital hypophosphatemic rickets was therefore performed, revealing heterozygous ENPP1 variants in cases 1 and 2 (case 1, c.536A>G, p.Asn179Ser; case 2, c.1352A>G, p.Tyr451Cys) and compound heterozygous ENPP1 variants in case 3 constituting the same variants present in cases 1 and 2 (c.536A>G, p.Asn179Ser and c.1352A>G, p.Tyr451Cys). Several in silico tools predicted the two variants to be pathogeneic, a finding confirmed by in vitro biochemical analysis demonstrating that the p.Asn179Ser and p.Tyr451Cys ENPP1 variants possessed a catalytic velocity of 45% and 30% compared with that of wild-type ENPP1, respectively. Both variants were therefore categorized as pathogenic loss-of-function mutations. Our findings suggest that ENPP1 mutational status should be evaluated in patients presenting with the diagnosis of idiopathic DISH, ossification of the posterior longitudinal ligament (OPLL), and early-onset osteoporosis. © 2022 American Society for Bone and Mineral Research (ASBMR).

Cardiomyocytes disrupt pyrimidine biosynthesis in nonmyocytes to regulate heart repair.

Various populations of cells are recruited to the heart after cardiac injury, but little is known about whether cardiomyocytes directly regulate heart repair. Using a murine model of ischemic cardiac injury, we demonstrate that cardiomyocytes play a pivotal role in heart repair by regulating nucleotide metabolism and fates of nonmyocytes. Cardiac injury induced the expression of the ectonucleotidase ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which hydrolyzes extracellular ATP to form AMP. In response to AMP, cardiomyocytes released adenine and specific ribonucleosides that disrupted pyrimidine biosynthesis at the orotidine monophosphate (OMP) synthesis step and induced genotoxic stress and p53-mediated cell death of cycling nonmyocytes. As nonmyocytes are critical for heart repair, we showed that rescue of pyrimidine biosynthesis by administration of uridine or by genetic targeting of the ENPP1/AMP pathway enhanced repair after cardiac injury. We identified ENPP1 inhibitors using small molecule screening and showed that systemic administration of an ENPP1 inhibitor after heart injury rescued pyrimidine biosynthesis in nonmyocyte cells and augmented cardiac repair and postinfarct heart function. These observations demonstrate that the cardiac muscle cell regulates pyrimidine metabolism in nonmuscle cells by releasing adenine and specific nucleosides after heart injury and provide insight into how intercellular regulation of pyrimidine biosynthesis can be targeted and monitored for augmenting tissue repair.

Musculoskeletal Comorbidities and Quality of Life in ENPP1-Deficient Adults and the Response of Enthesopathy to Enzyme Replacement Therapy in Murine Models.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) deficiency leads to cardiovascular calcification in infancy, fibroblast growth factor 23 (FGF23)-mediated hypophosphatemic rickets in childhood, and osteomalacia in adulthood. Excessive enthesis mineralization and cervical spine fusion have been previously reported in patients with biallelic ENPP1 deficiency, but their effect on quality of life is unknown. We describe additional musculoskeletal complications in patients with ENPP1 deficiency, namely osteoarthritis and interosseous membrane ossification, and for the first time evaluate health-related quality of life (HRQoL) in patients with this disease, both subjectively via narrative report, and objectively via the Brief Pain Inventory-Short Form, and a Patient Reported Outcome Measurement Information System Physical Function (PROMIS PF) short form. Residual pain, similar in magnitude to that identified in adult patients with X-linked hypophosphatemia, was experienced by the majority of patients despite use of analgesic medications. Impairment in physical function varied from mild to severe. To assess murine ENPP1 deficiency for the presence of enthesopathy, and for the potential response to enzyme replacement therapy, we maintained Enpp1asj/asj mice on regular chow for 23 weeks and treated cohorts with either vehicle or a long-acting form of recombinant ENPP1. Enpp1asj/asj mice treated with vehicle exhibited robust calcification throughout their Achilles tendons, whereas two-thirds of those treated with ENPP1 enzyme replacement exhibited complete or partial suppression of the Achilles tendon calcification. Our combined results document that musculoskeletal complications are a significant source of morbidity in biallelic ENPP1 deficiency, a phenotype which is closely recapitulated in Enpp1asj/asj mice. Finally, we show that a long-acting form of recombinant ENPP1 prevents the development of enthesis calcification at the relatively modest dose of 0.3 mg/kg per week, suggesting that suppression of enthesopathy may be attainable upon dose escalation. © 2021 American Society for Bone and Mineral Research (ASBMR). This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Direct targeting of amplified gene loci for proapoptotic anticancer therapy.

Gene amplification drives oncogenesis in a broad spectrum of cancers. A number of drugs have been developed to inhibit the protein products of amplified driver genes, but their clinical efficacy is often hampered by drug resistance. Here, we introduce a therapeutic strategy for targeting cancer-associated gene amplifications by activating the DNA damage response with triplex-forming oligonucleotides (TFOs), which drive the induction of apoptosis in tumors, whereas cells without amplifications process lower levels of DNA damage. Focusing on cancers driven by HER2 amplification, we find that TFOs targeting HER2 induce copy number-dependent DNA double-strand breaks (DSBs) and activate p53-independent apoptosis in HER2-positive cancer cells and human tumor xenografts via a mechanism that is independent of HER2 cellular function. This strategy has demonstrated in vivo efficacy comparable to that of current precision medicines and provided a feasible alternative to combat drug resistance in HER2-positive breast and ovarian cancer models. These findings offer a general strategy for targeting tumors with amplified genomic loci.

Response of the ENPP1-Deficient Skeletal Phenotype to Oral Phosphate Supplementation and/or Enzyme Replacement Therapy: Comparative Studies in Humans and Mice.

Inactivating mutations in human ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) may result in early-onset osteoporosis (EOOP) in haploinsufficiency and autosomal recessive hypophosphatemic rickets (ARHR2) in homozygous deficiency. ARHR2 patients are frequently treated with phosphate supplementation to ameliorate the rachitic phenotype, but elevating plasma phosphorus concentrations in ARHR2 patients may increase the risk of ectopic calcification without increasing bone mass. To assess the risks and efficacy of conventional ARHR2 therapy, we performed comprehensive evaluations of ARHR2 patients at two academic medical centers and compared their skeletal and renal phenotypes with ENPP1-deficient Enpp1asj/asj mice on an acceleration diet containing high phosphate treated with recombinant murine Enpp1-Fc. ARHR2 patients treated with conventional therapy demonstrated improvements in rickets, but all adults and one adolescent analyzed continued to exhibit low bone mineral density (BMD). In addition, conventional therapy was associated with the development of medullary nephrocalcinosis in half of the treated patients. Similar to Enpp1asj/asj mice on normal chow and to patients with mono- and biallelic ENPP1 mutations, 5-week-old Enpp1asj/asj mice on the high-phosphate diet exhibited lower trabecular bone mass, reduced cortical bone mass, and greater bone fragility. Treating the Enpp1asj/asj mice with recombinant Enpp1-Fc protein between weeks 2 and 5 normalized trabecular bone mass, normalized or improved bone biomechanical properties, and prevented the development of nephrocalcinosis and renal failure. The data suggest that conventional ARHR2 therapy does not address low BMD inherent in ENPP1 deficiency, and that ENPP1 enzyme replacement may be effective for correcting low bone mass in ARHR2 patients without increasing the risk of nephrocalcinosis. © 2021 American Society for Bone and Mineral Research (ASBMR).

Genetic pathways disrupted by ENPP1 deficiency provide insight into mechanisms of osteoporosis, osteomalacia, and paradoxical mineralization.

Ectonucleotide phosphatase/phosphodiesterase 1 (ENPP1) deficiency results in either lethal arterial calcifications ('Generalized Arterial Calcification of Infancy' - GACI), phosphate wasting rickets ('Autosomal Recessive Hypophosphatemic Rickets type 2' - ARHR2), early onset osteoporosis, or progressive spinal rigidity ('Ossification of the Posterior Longitudinal Ligament' - OPLL). As ENPP1 generates a strong endogenous mineralization inhibitor - extracellular pyrophosphate (PPi) - ENPP1 deficiency should not result in reduced bone volume, and therefore the mechanism ENPP1 associated osteoporosis is not apparent given current understanding of the enzyme's function. To investigate genetic pathways driving the skeletal phenotype of ENPP1 deficiency we compared gene expression in Enpp1asj/asj mice and WT sibling pairs by RNAseq and qPCR in whole bones, and in the liver and kidney by qPCR, directly correlating gene expression with measures of bone microarchitectural and biomechanical phenotypes. Unbiased analysis of the differentially expressed genes compared to relevant human disease phenotypes revealed that Enpp1asj/asj mice exhibit strong signatures of osteoporosis, ARHR2 and OPLL. We found that ENPP1 deficient mice exhibited reduced gene transcription of Wnt ligands in whole bone and increased transcription of soluble Wnt inhibitors in the liver and kidney, suggestive of multiorgan inhibition of Wnt activity. Consistent with Wnt suppression in bone, Collagen gene pathways in bone were significantly decreased and Fgf23 was significantly increased, all of which directly correlated with bone microarchitectural defects and fracture risk in Enpp1asj/asj mice. Moreover, the bone findings in 10-week old mice correlated with Enpp1 transcript counts but not plasma [PPi], suggesting that the skeletal phenotype at 10 weeks is driven by catalytically independent ENPP1 function. In contrast, the bone findings in 23-week Enpp1asj/asj mice strongly correlated with plasma PPi, suggesting that chronically low PPi drives the skeletal phenotype in older mice. Finally, correlation between Enpp1 and Fgf23 transcription suggested ENPP1 regulation of Fgf23, which we confirmed by dosing Enpp1asj/asj mice with soluble ENPP1-Fc and observing suppression of intact plasma FGF23 and ALP. In summary, our findings suggest that osteoporosis associated with ENPP1 deficiency involves the suppression of Wnt via catalytically independent Enpp1 pathways, and validates Enpp1asj/asj mice as tools to better understand OPLL and Paradoxical Mineralization Disorders.

Improving the Pharmacodynamics and In Vivo Activity of ENPP1-Fc Through Protein and Glycosylation Engineering.

Enzyme replacement with ectonucleotide pyrophosphatase phospodiesterase-1 (ENPP1) eliminates mortality in a murine model of the lethal calcification disorder generalized arterial calcification of infancy. We used protein engineering, glycan optimization, and a novel biomanufacturing platform to enhance potency by using a three-prong strategy. First, we added new N-glycans to ENPP1; second, we optimized pH-dependent cellular recycling by protein engineering of the Fc neonatal receptor; finally, we used a two-step process to improve sialylation by first producing ENPP1-Fc in cells stably transfected with human α-2,6-sialyltransferase (ST6) and further enhanced terminal sialylation by supplementing production with 1,3,4-O-Bu3 ManNAc. These steps sequentially increased the half-life of the parent compound in rodents from 37 hours to ~ 67 hours with an added N-glycan, to ~ 96 hours with optimized pH-dependent Fc recycling, to ~ 204 hours when the therapeutic was produced in ST6-overexpressing cells with 1,3,4-O-Bu3 ManNAc supplementation. The alterations were demonstrated to increase drug potency by maintaining efficacious levels of plasma phosphoanhydride pyrophosphate in ENPP1-deficient mice when the optimized biologic was administered at a 10-fold lower mass dose less frequently than the parent compound-once every 10 days vs. 3 times a week. We believe these improvements represent a general strategy to rationally optimize protein therapeutics.

Unraveling the mechanism of recognition of the 3' splice site of the adenovirus major late promoter intron by the alternative splicing factor PUF60.

Pre-mRNA splicing is critical for achieving required amounts of a transcript at a given time and for regulating production of encoded protein. A given pre-mRNA may be spliced in many ways, or not at all, giving rise to multiple gene products. Numerous splicing factors are recruited to pre-mRNA splice sites to ensure proper splicing. One such factor, the 60 kDa poly(U)-binding splicing factor (PUF60), is recruited to sites that are not always spliced, but rather function as alternative splice sites. In this study, we characterized the interaction of PUF60 with a splice site from the adenovirus major late promoter (the AdML 3' splice site, AdML3'). We found that the PUF60-AdML3' dissociation constants are in the micromolar range, with the binding affinity predominantly provided by PUF60's two central RNA recognition motifs (RRMs). A 1.95 Å crystal structure of the two PUF60 RRMs in complex with AdML3' revealed a dimeric organization placing two stretches of nucleic acid tracts in opposing directionalities, which can cause looping of nucleic acid and explain how PUF60 affects pre-mRNA geometry to effect splicing. Solution characterization of this complex by light-scattering and UV/Vis spectroscopy suggested a potential 2:1 (PUF602:AdML3') stoichiometry, consistent with the crystal structure. This work defines the sequence specificity of the alternative splicing factor PUF60 at the pre-mRNA 3' splice site. Our observations suggest that control of pre-mRNA directionality is important in the early stage of spliceosome assembly, and advance our understanding of the molecular mechanism by which alternative and constitutive splicing factors differentiate among 3' splice sites.

Next generation miRNA inhibition using short anti-seed PNAs encapsulated in PLGA nanoparticles.

Selective inhibition of microRNAs (miRNAs) offers a new avenue for cancer therapeutics. While most of the current anti-miRNA (antimiR) reagents target full length miRNAs, here we investigate novel nanoparticle-delivered short PNA probes containing cationic domains targeting the seed region of the miRNA for effective antimiR therapy. For proof of concept, we tested PNAs targeting miRNA-155 and employed poly(lactic-co-glycolic acid) (PLGA)-based nanoparticle formulation for delivery. A comprehensive evaluation of PLGA nanoparticles (NPs) containing short PNA probes showed significantly superior loading, release profile, and uniform size distribution, compared to conventional non-cationic PNA probes. Confocal microscopy and flow cytometry analyses showed efficient transfection efficiency and uniform distribution of PLGA NPs containing short PNA probes in the cytoplasm. Functional analysis also confirmed efficient miRNA-155 inhibition including an effect on its downstream target proteins. Further, reduced tumor growth was observed after systemic delivery of PLGA nanoparticles containing short PNA probes in vivo in a xenograft mouse model following inhibition of miR-155. There was no evidence of acute or chronic toxicity associated with systemic delivery of PLGA NPs containing short PNA probes in the mice. Overall, in this paper we present a novel antimiR strategy based on PLGA nanoparticle delivered short PNA probes for potential cancer therapy.

Phylogeny and chemistry of biological mineral transport.

Three physiologically mineralizing tissues - teeth, cartilage and bone - have critical common elements and important evolutionary relationships. Phylogenetically the most ancient densely mineralized tissue is teeth. In jawless fishes without skeletons, tooth formation included epithelial transport of phosphates, a process echoed later in bone physiology. Cartilage and mineralized cartilage are skeletal elements separate from bone, but with metabolic features common to bone. Cartilage mineralization is coordinated with high expression of tissue nonspecific alkaline phosphatase and PHOSPHO1 to harvest available phosphate esters and support mineralization of collagen secreted locally. Mineralization in true bone results from stochastic nucleation of hydroxyapatite crystals within the cross-linked collagen fibrils. Mineral accumulation in dense collagen is, at least in major part, mediated by amorphous aggregates - often called Posner clusters - of calcium and phosphate that are small enough to diffuse into collagen fibrils. Mineral accumulation in membrane vesicles is widely suggested, but does not correlate with a definitive stage of mineralization. Conversely mineral deposition at non-physiologic sites where calcium and phosphate are adequate has been shown to be regulated in large part by pyrophosphate. All of these elements are present in vertebrate bone metabolism. A key biological element of bone formation is an epithelial-like cellular organization which allows control of phosphate, calcium and pH during mineralization.

Human Heterozygous ENPP1 Deficiency Is Associated With Early Onset Osteoporosis, a Phenotype Recapitulated in a Mouse Model of Enpp1 Deficiency.

Biallelic ENPP1 deficiency in humans induces generalized arterial calcification of infancy (GACI) and/or autosomal recessive hypophosphatemic rickets type 2 (ARHR2). The latter is characterized by markedly increased circulating FGF23 levels and renal phosphate wasting, but aberrant skeletal manifestations associated with heterozygous ENPP1 deficiency are unknown. Here, we report three adult men with early onset osteoporosis who presented with fractures in the thoracic spine and/or left radius, mildly elevated circulating FGF23, and hypophosphatemia. Total hip bone mineral density scans demonstrated osteoporosis (Z-score < -2.5) and HRpQCT demonstrated microarchitectural defects in trabecular and cortical bone. Next-generation sequencing revealed heterozygous loss-of-function mutations in ENPP1 previously observed as biallelic mutations in infants with GACI. In addition, we present bone mass and structure data as well as plasma pyrophosphate (PPi) data of two siblings suffering from ARHR2 in comparison to their heterozygous and wild-type family members indicative of an ENPP1 gene dose effect. The skeletal phenotype in murine Enpp1 deficiency yielded nearly identical findings. Ten-week-old male Enpp1 asj/asj mice exhibited mild elevations in plasma FGF23 and hypophosphatemia, and micro-CT analysis revealed microarchitectural defects in trabecular and cortical bone of similar magnitude to HRpQCT defects observed in humans. Histomorphometry revealed mild osteomalacia and osteopenia at both 10 and 23 weeks. The biomechanical relevance of these findings was demonstrated by increased bone fragility and ductility in Enpp1 asj/asj mice. In summary, ENPP1 exerts a gene dose effect such that humans with heterozygous ENPP1 deficiency exhibit intermediate levels of plasma analytes associated with bone mineralization disturbance resulting in early onset osteoporosis. © 2019 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.