JBMR PlusVolume 2, Issue S2 p. S1-S14 Society AbstractsOpen Access Abstracts from the First Scientific Meeting of the Soft Bones Foundation First published: 22 December 2018 https://doi.org/10.1002/jbm4.10082AboutSectionsPDF 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 Share a linkShare onFacebookTwitterLinkedInRedditWechat Foreword From June 8 to 10, 2018, in Chicago, IL, USA, a multinational group of 50 clinicians, clinical investigators, and basic scientists gathered with other interested individuals to convene the First Scientific Meeting of the Soft Bones Foundation. They reviewed current understanding and uncertainties concerning hypophosphatasia (HPP) and planned work necessary to improve the lives of people with this inborn error of metabolism. The importance for such a meeting was perceived by Ms. Deborah Fowler, President and Founder of the Soft Bones Foundation, and was brought to success with the help of Ms. Denise Goodbar and Ms. Charlene Waldman and support from Alexion Pharmaceuticals, PANTHERx Specialty Pharmacy, and Charles River Laboratories, Inc. Session moderators Matthew Drake, MD, PhD, Eric Rush, MD, Frank Rauch, MD, Mark Nunes, MD, Kathryn Dahir, MD, Craig Langman, MD, and Susan Ott, MD, expertly kept to time the considerable enthusiasm of all attendees. Graham Russell, MD, PhD, gave a historical review of inorganic pyrophosphate, its action as an inhibitor of mineralization, excess in HPP, and early experiences following its modification to become the bisphosphonates. Michael Whyte, MD, described the discovery in 1923 of alkaline phosphatase (ALP), the first report in 1948 of HPP, and the many important lessons from investigation of patients, including major insight concerning the pathogenesis of the defective hard tissue mineralization leading to tooth loss and rickets during childhood and osteomalacia during adult life as well as identification of its etiology. The expanded clinical nosology of HPP was validated after using the now delineated clinical, biochemical, radiological, and histopathological features of this metabolic bone disease. Stephen Coburn, PhD, discussed the derangement of vitamin B6 metabolism leading to extracellular accumulation of the deficient tissue-nonspecific ALP isoenzyme (TNSALP) substrates including pyridoxal 5′-phosphate, and the pathogenesis of the vitamin B6-dependent seizures. Jose Luis Millan, PhD, reviewed the importance of mouse models for HPP, including their role in preclinical studies of asfotase alfa (AA) enzyme-replacement therapy for HPP, and investigation of other molecules that regulate skeletal mineralization. Larry Suva, PhD, described the recent development of the first large animal model (ie, sheep) for HPP, and the early findings concerning the clinical and biochemical phenotype. Deborah Kraków, MD, discussed the considerable uncertainties with prenatal radiological imaging of HPP, and Deborah Wenkert, MD, defined and reviewed the management of the not uncommon “benign prenatal” form. Steven Mumm, PhD, provided an overview concerning the Mendelian inheritance of HPP, and what can be said concerning genotype/phenotype correlations. Treatment for HPP now includes the benefits from enzyme replacement using AA (Strensiq™), approved multinationally in 2015 typically for pediatric-onset HPP. Philippe Crine, PhD, reviewed the recombinant DNA structuring of AA as a TNSALP targeted to hydroxyapatite. Jill Simmons, MD, described the improvements in skeletal mineralization, respiratory and motor function, and growth observed during AA treatment studied initially for the life-threatening perinatal and infantile forms of HPP and followed to 7 years of therapy. Nicholas Bishop, MD, and Vrinda Saraff, MD, reported experience with such patients in the United Kingdom. Keiichi Ozono, MD, PhD, presented the Japanese experience with severe pediatric HPP. Then, Gary Gottesman, MD, provided an overview concerning AA treatment for older children debilitated by HPP, including the problems as well as the benefits. Donna Griffin, PT, PCS, reported how a modification of the Performance-Oriented Mobility Assessment-gait (mPOMA-G) could assess in real-time baseline and treatment responses of children with HPP. Lothar Seefried, MD, discussed the orthopedic complications and management of adults with HPP, including experience with AA treatment. The broad range of HPP severity encountered in this age group was reviewed by Peter Tebben, MD, and Frederick Singer, MD, discussed the difficulties with diagnosis and treatment of these individuals. Priya Krishnani, MD, outlined the importance of functional testing of adults with HPP, and what improvements have been observed with AA treatment. Pauline Camacho, MD, discussed “off label” use of parathyroid hormone given to adults with HPP. Mark Rallo, OD, discussed occurrences of ectopic calcification on conjunctiva naturally in HPP, and perhaps increased with AA treatment but largely microscopic and asymptomatic. Timothy Wright, MS, DDS, reviewed oral and craniofacial issues of HPP, and emphasized how much more must be learned about these complications, especially in adults. Brian Foster, PhD, discussed the importance of TNSALP in dentoalveolar tissue formation, and the consequences of TNSALP deficiency in HPP. Clifford Rosen, MD, reviewed early work concerning a potential role for ALP in fat metabolism, including fat accumulation at sites of AA injection in patients with HPP. Isabel Orriss, PhD, provided an overview of skeletal mineralization controlled by TNSALP and pyrophosphate, and other factors. Progress reports, summarized in their abstracts compiled herein, were provided by the past recipients of research awards from the Soft Bones Foundation: Steven Mumm, PhD, Luke Mortensen, PhD, Brian Foster, PhD, and Katheryn Dahir, MD. Finally, means to advance the treatment or to cure HPP, including by gene editing, ALP transfection, and mesenchymal stem cell therapy, were discussed by José Luis Millan, PhD, and Luke Mortensen, PhD. Here are the abstracts from the meeting. We are grateful to JBMR Plus for publishing them, so that they can be accessed worldwide. Michael P. Whyte, MD Chair, Scientific Advisory Board Soft Bones Foundation Boonton, NJ, USA https://www.softbones.org/ The identification of inorganic pyrophosphate (PPi) as a key regulator of biological calcification can be traced back to the 1960s, starting with the work of Fleisch and colleagues.1 Hypophosphatasia (HPP) was identified as the first of the pyrophosphate diseases.2,3 This established the notion that ALP was an important enzyme in regulating extracellular pyrophosphate levels. In the early years, we established that pyrophosphate appearing in blood and urine was endogenous rather than dietary in origin. Studies with 32P-labeled PPi showed that it had a relatively high turnover in the extracellular compartment.4 PPi underwent intrarenal hydrolysis, a process that could be inhibited by phosphate, at that time being considered as a potential treatment for urolithiasis. There are very many enzyme reactions involved in biosynthesis that generate huge amounts (kg/day) of pyrophosphate within cells. However, it is now recognized that pyrophosphate in body fluids is generated and destroyed at cell surfaces by the combined actions of ectonucleotide pyrophosphatase/phosphodiesterase (ENPP) and ALP. The role of the ANK transport system in facilitating the movement of PPi across cell membranes remains unclear, although mutations in ANK have been implicated in the pathogenesis of chondrocalcinosis.5 Attempts to use pyrophosphate or polyphosphates to prevent pathological calcification were thwarted by its rapid hydrolysis in the body and its failure to be absorbed across the intestinal tract. It was these observations that prompted studies of bisphosphonates as pyrophosphate analogues. The discovery of the biological effects of bisphosphonates (BPs), especially as inhibitors of bone resorption, led to their eventual and still current successful use as the major drugs for the treatment of bone resorption disorders, including Paget's disease, bone metastases, and osteoporosis.6 References 1 Fleisch, H, Russell, RGG, Straumann, F. Effect of pyrophosphate on hydroxyapatite and its implications in calcium homeostasis. Nature. 1966; 212:901–3. 2 Russell, RGG. Excretion of inorganic pyrophosphate in hypophosphatasia. Lancet. 1965;2(7410):461–4. 3 Russell, RGG, Bisaz, S, Donath, A, Morgan, DB, Fleisch, H. Inorganic pyrophosphate in plasma in normal persons and in patients with hypophosphatasia, osteogenesis imperfecta and other disorders of bone. J Clin Invest. 1971;50(5):961–9. 4 Jung, A, Russell, RGG, Bisaz, S, Morgan, DB, Fleisch, H. (1970) The fate of intravenously injected 32P-pyrophosphate in dogs. American Journal of Physiology, 1970;218(6):1757–64. 5 Orriss, IR, Arnett, TR, Russell, RGG. Pyrophosphate: a key inhibitor of mineralisation. Curr Opin Pharmacol. 2016;28:57–68. 6 Russell, RGG. Bisphosphonates: the first 40 years. Bone. 2011;49:2–19. Although hypophosphatasia (HPP) is rare, manifests the greatest severity range of all skeletal diseases, and was the last rickets/osteomalacia to acquire a medical treatment, most of what we know about the function of ALP has come from studies of HPP patients. In 1948, JC Rathbun coined “hypophosphatasia” to describe the lethal rickets and epilepsy of an infant paradoxically lacking serum and skeletal ALP activity. Soon after, premature deciduous tooth loss from hypomineralized cementum extended the pathogenesis to involve both hard tissues. Elevations in HPP urine of phosphoethanolamine (PEA) and inorganic pyrophosphate (PPi) and also pyridoxal 5′-phosphate (PLP) in blood would disclose each to be a natural substrate for ALP. Amino acid sequence analysis of proteolytic digests of ALP purified from HPP autopsy tissues indicated HPP is an inborn error of the tissue nonspecific isoenzyme of ALP (TNSALP). Years later, mutation analysis confirmed that all HPP patients carry one or two defective (TNSALP) alleles. This finding verified, after its discovery in 1923, that ALP acts in skeletal mineralization. The disrupted metabolism of vitamin B6 featuring elevated circulating PLP indicated TNSALP is a cell-surface protein. Soon after, phosphatidylinositol-glycan was discovered to link ALPs and other proteins to cell surfaces and contain PEA. Vitamin B6–dependent seizures reflect especially severe HPP by diminishing neurotransmitter biosynthesis co-factored by PLP in the brain. The culprit for the defective mineralization was shown to be PPi. Nascent hydroxyapatite (HA) crystals form in HPP matrix vesicles (MVs), but fail to grow after MVs rupture. Correction of PEA, PLP, and PPi accumulation in HPP carrier women near the end of pregnancy (when placental ALP circulates transiently reversing their hypophosphatasemia) indicated the four ALP isoenzymes in humans share a similar catalytic domain. The hyperphosphatemia characteristic of HPP indicates that TNSALP somehow promotes urinary excretion of inorganic phosphate (Pi). Now, the extraordinary severity range of HPP is explained by >340, often missense, mutations identified throughout the catalytically active homodimeric form of TNSALP. In 2015, successful asfotase alfa treatment for HPP patients “closed the loop,” verifying TNSALP's role in biomineralization in humans. Decreased activity of tissue nonspecific alkaline phosphatase (TNSALP) in hypophosphatasia (HPP) is associated with markedly increased concentrations of pyridoxal 5′-phosphate (PLP) in plasma. The plasma concentrations of pyridoxal (PL) and 4-pyridoxic acid remain normal in HPP, which distinguishes it from increased dietary intake of vitamin B6, which raises the plasma concentrations of all three vitamers. Organ ablation studies in dogs demonstrated that the liver is the primary source of plasma PLP, probably secreted associated with albumin. The half-life of a 5 mg i.v. dose of PLP in normal humans is 2 to 3 hours. Plasma PLP concentrations normally do not exceed 1000 nM, presumably reflecting maximal production by the liver. During pregnancy in carriers of HPP plasma activity of placental ALP increases accompanied by a decline in plasma PLP concentration. PLP increases after delivery. Although TNSALP is an exoenzyme, there appears to be a specific PLP phosphatase within the cell. As a result, there is little evidence of altered intracellular metabolism of vitamin B6 in humans with HPP other than pyridoxine-dependent seizures in some neonatal and infant cases. Data from knockout mice suggest the seizures result from decreased gamma-aminobutyrate. In these mice i.p. PL was more effective than pyridoxine in prolonging survival. Although plasma PL values remain normal in HPP patients, plasma PL and tissue B6 vitamer concentrations are reduced in the knockout mice. Clinical assays for ALP usually use nonphysiological conditions and substrates. Under physiological conditions normal plasma concentrations of inorganic phosphate inhibit ALP about 50%. HPP may exhibit neuromuscular symptoms. About 75% of the vitamin B6 in the body is associated with glycogen phosphorylase in muscle. We have not found any data on glycogen phosphorylase activity in HPP. We are developing a physiologically based pharmacokinetic model of vitamin B6 metabolism that we hope will provide additional insights into vitamin B6 metabolism in normal and pathological conditions. Hypophosphatasia (HPP) is the heritable rare disease that results from ALPL gene mutations leading to deficient activity of the tissue-nonspecific alkaline phosphatase isozyme (TNAP). HPP primarily features rickets or osteomalacia and early loss of teeth. Alpl knockout mice (Alpl–/– aka Akp2–/– mice) phenocopy infantile HPP extremely well and have enabled us to probe mechanistically the role of TNAP in the pathophysiology of HPP. The mouse studies have proven that the skeletal and dental manifestations of HPP are primarily caused by the accumulation of extracellular inorganic pyrophosphate (PPi), a physiological substrate of TNAP and a potent mineralization inhibitor. Additionally, phosphorylated osteopontin (OPN), another potent mineralization inhibitor also accumulates, further restricting the degree of extracellular matrix mineralization. Severely affected HPP patients, as well as 100% of the Alpl–/– mice, suffer from severe seizures that herald a lethal outcome. The seizures are partly explained by inadequate availability of pyridoxal phosphate, another physiological substrate of TNAP, that is a cofactor in the synthesis of neurotransmitters in the central nervous system (CNS), but abnormal P2rX7 signaling in the CNS is also part of the mechanisms leading to seizures. Understanding this pathophysiology has provided the rationale for the current therapeutic intervention for pediatric onset HPP using recombinant mineral-targeted TNAP for enzyme replacement that dramatically restores survival, eliminates the seizures, and prevents the skeletal and dental phenotype of the treated Alpl–/– mice. The pathophysiology of other features of HPP (craniosynostosis, nephrocalcinosis, muscle weakness, inflammation, and pain), however, are not yet understood. Preliminary data emanating from the mouse models suggest that these manifestations may be caused at least in part by local changes in the ATP/adenosine ratio as a result of deficient hydrolysis of ATP, leading to altered purinergic signaling that affects cell behavior and tissue homeostasis. The utility of genome editing techniques provides a unique opportunity for musculoskeletal investigators to consider the examination of rare phenotypes in domestic animals, or perhaps more so, to develop domestic animal models of bone disorders in which the bone remodeling process more closely resembles that observed in humans. Indeed, an ideal animal model of a human disease should satisfy several criteria: (i) the model should closely approximate the condition under investigation; (ii) the model should allow longitudinal study and permit appropriate tests to be performed; and (iii) specific analytical reagents should be available. To date, the use of murine models has been driven almost exclusively by the availability of mouse genetics and their minimal animal costs; the ability to manipulate the rodent genome, the short reproductive cycles, and the generation of a range of genetically identical inbred strains of mice has forced investigators down the murine route. However, unlike humans, rodents have smaller long bones with thin fragile cortices that lack Haversian remodeling, which is the fundamental process by which larger animals, such as sheep and humans, model and remodel their skeletons throughout life. To accurately model the associated musculoskeletal and dental phenotypes of a rare human bone disease, hypophosphatasia (HPP), we utilized domestic sheep as a new animal model. Using gene editing tools, a single point mutation was introduced into the tissue nonspecific alkaline phosphatase (TNSALP) gene (ALPL) (1077C>G), generating the first large animal model of HPP. Compared to wild-type (WT) controls, HPP sheep have significantly reduced serum alkaline phosphatase activity, decreased tail vertebral bone size, and metaphyseal flaring, consistent with mineralization deficits observed in human HPP patients. Additionally, unlike mice that are monophyodont (one set of teeth), sheep and humans are diphyodont (two sets of teeth), including primary and secondary sets. Oral radiographs and computed tomography revealed thin dentin and wide pulp chambers in incisors, and radiolucency of mandibular bone in HPP versus WT sheep, accurately replicating odonto-HPP. Skeletal muscle biopsies revealed aberrant fiber size and mitochondrial cristae structure in HPP versus WT sheep. These genetically engineered sheep provide opportunities to follow HPP progression longitudinally, allowing repeated bone and muscle biopsies, timed administration of treatment(s), evaluation of primary and secondary tooth development and retention, and long-term assessment of periodontal health and interventions such as orthodontics and dental implants, all of which are the focus of ongoing investigation in our laboratory. The incidence of congenital birth defects is 3% to 5% in the general population. Among those are the skeletal dysplasias which account for 5% of those, or 0.15% of all birth defects. The skeletal dysplasias are a heterogeneous group of more than 450 distinct disorders, many of which present in the prenatal period. Among those, hypophosphatasia (HPP) can be inherited as both a recessive or dominant disorder. The prenatal form of HPP is severe, often perinatal lethal, and results from biallelic mutations in the gene that encodes tissue nonspecific alkaline phosphatase (TNSALP). Prenatal detected cases are usually detected by screening ultrasounds, often in families with no known histories of genetic skeletal disorders. Prenatal findings in the perinatal form of HPP include poor mineralization of the calvarium, short and fragmented appendicular bones, metaphyseal irregularities, bending and fractures (including the ribs), short wavy ribs, hypomineralization of the scapula, flattened spine with irregular mineralization and poor mineralization of the distal elements of the appendicular skeleton, and polyhydramnios later in gestation. Although postnatal radiographs are usually diagnostic, prenatal findings can overlap with severe forms of osteogenesis imperfecta, campomelic dysplasia, bent bone-FGFR2 type, and other bent bone skeletal dysplasias. Increasing employment of carrier screening of at risk families (preconception or pregnancy) may lead to earlier and specific detection of HPP, providing more tailored care. In 2011, we published 17 new cases of “benign” prenatal hypophosphatasia (BP-HPP), 10% of our pediatric HPP population, and reviewed 24 previously published patients. These patients typically improve during the third trimester (when placental ALP rises and ALP substrates in a carrier mother fall) as well as after birth. The highly variable clinical and biochemical characteristics ex utero, however, are better aligned with a reclassification based on the age and severity of their first additional ex utero clinical manifestation(s). We showed that the following does NOT predict lethality: Recessive inheritance (AR) ∘ 9/17 of our cohort and 16 or 17/24 in the literature had AR-BP-HPP Severe in utero skeletal demineralization ∘ Seen in 3/8 of our cohort (where mineralization was ascertained) and one literature patient including one with typical perinatal skull and spine demineralization abnormalities. Bowdler's spurs ∘ Patients in the literature and one of our patients had a Bowdler spur (14/16 had lower leg skin dimples). In utero bowing or shortening of long bones ∘ 11/17 of our cohort had bowed and shortened extremities ∘ 2/17 short without bowing, 4/17 bowed without shortening. Multiple affected limbs in utero ∘ Only 1/ 17 had only a single affected limb (femur) Early gestational age of abnormalities ∘ 2/17 had a skeletal dysplasia at ∼13 weeks gestation Combination of the above ∘ All three in our cohort with poor mineralization had abnormal ultrasounds at 14 to 15 weeks gestation with multiple bones affected and tibial dimples. Two had AR-BP-HPP. Sibling with HPP who did not have in utero manifestations ∘ Three HPP sibships had similar ex utero manifestations but discordance for BP-HPP. ∘ In one additional discordant sibship, a girl with childhood AD-BP-HPP had a sister with no in utero manifestations but AR infantile HPP. Preterm evaluation ∘ An identical single TNAP mutation and similar severe prenatal HPP were found early in two pregnancies. The fetal autopsy on the terminated pregnancy concluded that the HPP was incompatible with life. His brother significantly improved during the third trimester and has childhood AD-BP-HPP. The following prenatal findings may be somewhat reassuring: A normal chest shape, chest circumference and/or abdominal circumference ∘ Noted in 11/17 of our cohort ∘ No related abnormalities were noted in our cohort nor in those in the literature AD inheritance Maternal TNAP mutation, although not predictive of survivability ∘ True for 16/17 in our cohort and likely for 23/24 in the literature. Hypophosphatasia (HPP) is caused by loss-of-function mutations in ALPL, the gene encoding the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP). HPP's extraordinarily broad range of severity from early tooth loss without skeletal disease to complete lack of skeletal mineralization is now being largely explained by the number and nature of ALPL mutations. Inheritance can be either dominant, caused by a single ALPL defect generally associated with mild disease, or recessive, caused by two ALPL mutations and generally showing moderate to severe disease. In HPP, 355 different mutations have been reported and include missense, nonsense, and splice site defects, and a range of insertions and deletions involving a single nucleotide up to multiple exon deletions. A single mutation has been reported in the 5′ untranslated region (UTR) at the major transcription start site. These ALPL/HPP defects are curated in an online database (The Tissue Nonspecific Alkaline Phosphatase Gene Mutations Database; http://www.sesep.uvsq.fr/03_hypo_mutations.php). The large majority (∼75%) are missense and are spread across the 524 amino acid protein, changing about 200 different amino acids. In our cohort of >300 probands, there are two common dominant and several common recessive mutations that likely represent founder events. The most common dominant mutation (Asp378Val) in America usually shows a mild phenotype when inherited singly, but has been severe when accompanied by a second defect. Common recessive defects (Glu191Lys, Ala176Thr, and Asp294Ala, respectively) show increasing disease severity, semi-independently of the second mutation. Residual TNSALP enzymatic activity of individual mutations, assessed using in vitro transfection studies, reportedly correlates with HPP disease severity. 3D molecular modeling shows that many severe mutations affect crucial domains of the homodimeric enzyme structure, e.g., the active site. TNSALP protein instability and cell localization studies demonstrating inability of some mutant proteins to reach the cell surface also explain some phenotypic severity. The phenotype of untreated HPP generally remains stable throughout childhood, but sometimes appears to progress throughout adulthood. This is an active area of our research, focusing on the two common dominant American mutations (Asp378Val and Asn417Ser). Asfotase alfa is a hybrid molecule engineered to contain recombinant human soluble tissue-nonspecific alkaline phosphatase (TNSALP), the constant region of human IgG1 Fc domain (Fc) and a deca-aspartate motif (D10). Purified asfotase alfa binds to hydroxyapatite mineral in vitro 32-fold more efficiently than does the bovine kidney TNSALP. Mineral-bound asfotase alfa remains fully active. The pharmacokinetics (PK) and tissue distribution of asfotase alfa in mice was established using a single intravenous bolus injection of 5 mg/kg. A circulating half-life of 34 hours was found, with prolonged retention of the [125I]-radiolabeled asfotase alfa in bones. Concentrations of the drug could be as high as 1 µg/g of bone (wet) weight in long bones such as femur and tibia. The total amount of radiolabeled asfotase alfa recovered in the whole skeleton was approximately 5% of the total injected. Skeletal levels of the bone-targeted material are stable, with no significant decrease in radiolabeled asfotase alfa observed after 4 days. No sustained accumulation of asfotase alfa was observed in muscle, where the amount of radiolabeled enzyme decreased in parallel with sALP-FcD10 in blood. In the absence of the deca-aspartate motif there was no preferential distribution of the compound in bone. As expected, the presence of the IgG1 Fc domain in the construct provided a very convenient purification first step due to its affinity to Protein A in chromatography matrices. Additional benefits of the presence of the Fc domain in the structure of asfotase alfa was to increase the circulating half-life and subcutaneous bioavailability compared to soluble TNSALP. Asfotase alfa was also found to be very stable even when formulated at high concentrations, making possible a subcutaneous route of injection in patients. In subsequent clinical trials this innovative engineered form of bone-targeted form of TSNALP proved to be of great benefit to hypophosphatasia patients. We have recently reported improved survival, skeletal manifestations, respiratory function, growth, and motor function in infants and young children with life-threatening HPP treated with asfotase alfa for up to 7 years.7 Skeletal manifestations were assessed with the Radiographic Global Impression of Change (RGI-C) scale (–3 = severe worsening; +3 = near/complete healing) and Rickets Severity Scale (RSS). Patient Z-scores evaluated growth. Motor and cognitive function were assessed using three scaled scores and patients transitioned between scales based upon age and functional ability: Bayley Scales of Infant Development III (BSID-III), Locomotion Subtest of the Peabody Developmental Motor Scales (PDMS-2), and Bruininks-Oseretsky Test of Motor Proficiency (BOT-2; Running Speed and Agility subtest). Score increases reflect greater skill acquisition. At baseline (BL), patients (n = 11) were median (minimum, maximum) 13.6 (0.7, 36.4) months of old. One patient withdrew consent; one died (sepsis unrelated to treatment).7 Nine of 11 patients received ≥6 years treatment and four of nine were treated for ≥7 years. RGI-C scores documented significant skeletal improvement by 3 months sustained through 7 years (p < 0.05). All evaluable patients demonstrated substantial healing (RGI-C ≥ 2) by year 4, sustained through year 7. RSS scores demonstrated similar improvement. Length/height and weight Z-scores from BL to year 7 reflected catch-up growth. BSID-III scaled scores at BL/first assessment indicated functional delay in nine of 11 patients. Median (minimum, maximum) Gross Motor BSID-III scaled scores improved from 1.0 (1, 8) at BL to 6.0 (2, 8) at year 3 (normative mean ± SD: 10 ± 3). Median Fine Motor and Cognitive BSID-III scaled scores were below normal at BL but normalized at years 2 and 3. Of eight patients who transitioned to PDMS-2, Locomotion standard scores increased during treatment, indicating motor skill acquisition, with standard scores within 1 SD of normal in five patients. Six patients transitioned to BOT-2 and had serial assessments; three achieved BOT-2 Running Speed and Agility scaled scores within normal range. There were no new safety concerns. The most common adverse events (AEs) were mild-to-moderate injection site reactions. I