Lentivirus-based HPSC therapy provides effective and long-term treatment in hypophosphatasia mouse model.

Bone Marrow (BM) Delivery of Genetically-Modified (gm) Adult CD34+ Hematopoietic Stem and Progenitor Cells (HSPC) Improves Homing and Engraftment of Short-Term Progenitors over Long-Term Repopulating Hematopoietic Stem Cells

210 - Analysis of the Functional Impact of RUNX1 Mutations in MDS Stem Cells

Lentiviral vector (LV)-mediated ex vivo gene therapy for haematopoietic stem and progenitor cells (HSPCs) has delivered on the promise of a 'one-and-done' treatment for several genetic diseases1. However, ex vivo manipulation and patient conditioning before transplantation are major hurdles that could be overcome by an in vivo approach. Here we demonstrate that in vivo gene delivery to HSPCs after systemic LV administration is enabled by the substantial trafficking of these cells from the liver to the bone marrow in newborn mice. We improved gene-transfer efficiency using a phagocytosis-shielded LV, successfully reaching bona fide HSPCs capable of long-term multilineage output and engraftment after serial transplantation, as confirmed by clonal tracking. HSPC mobilization further increased gene transfer, extending the window of intervention, although permissiveness to LV transduction declined with age. We successfully tested this in vivo strategy in mouse models of adenosine deaminase deficiency, autosomal recessive osteopetrosis and Fanconi anaemia. Interestingly, in vivo gene transfer provided a selective advantage to corrected HSPCs in Fanconi anaemia, leading to near-complete haematopoietic reconstitution and prevention of bone marrow failure. Given that circulating HSPCs in humans are also most abundant shortly after birth, in vivo HSPC gene transfer holds strong translational potential across multiple diseases.

A CD117-Amanitin Antibody Drug Conjugate (ADC) Effectively Depletes Human and Non-Human Primate Hematopoietic Stem and Progenitor Cells (HSPCs): targeted Non-Genotoxic Conditioning for Bone Marrow Transplant

A novel combination of biallelic ALPL mutations associated with adult hypophosphatasia: A phenotype-genotype association and computational analysis study.

Background. Hypophosphatasia (HPP) is caused by loss-of-function mutation(s) of the gene that encodes the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP). Consequently, cell-surface deficiency of TNSALP phosphohydrolase activity leads to extracellular accumulation of inorganic pyrophosphate, a natural substrate of TNSALP and inhibitor of mineralization. Children with HPP can manifest rickets, skeletal pain, deformity, fracture, muscle weakness, and premature deciduous tooth loss. Asfotase alfa is a recombinant, bone-targeted, human TNSALP injected s.c. to treat HPP. In 2012, we detailed the 1-year efficacy of asfotase alfa therapy for the life-threatening perinatal and infantile forms of HPP. Methods. Here, we evaluated the efficacy and safety of asfotase alfa treatment administered to children 6-12 years of age at baseline who were substantially impaired by HPP. Two radiographic scales quantitated HPP skeletal disease, including comparisons to serial radiographs from similarly affected historical control patients. Results. Twelve children receiving treatment were studied for 5 years. The 6-month primary endpoint was met, showing significant radiographic improvement. Additional significant improvements included patient growth, strength, motor function, agility, and quality of life, which for most patients meant achieving normal values for age- and sex-matched peers that were sustained at 5 years of treatment. For most, pain and disability resolved. Mild to moderate injection-site reactions were common and were sometimes associated with lipohypertrophy. Low anti-asfotase alfa antibody titers were noted in all patients. No evidence emerged for clinically important ectopic calcification or treatment resistance. Conclusions. Asfotase alfa enzyme replacement therapy has substantial and sustained efficacy with a good safety profile for children suffering from HPP. Trial Registration. ClinicalTrials.gov NCT00952484 (https://clinicaltrials.gov/ct2/show/NCT00952484) and NCT01203826 (https://clinicaltrials.gov/ct2/show/NCT01203826). Funding. Alexion Pharmaceuticals Inc. and Shriners Hospitals for Children.

32. Towards a Phase I Clinical Trial for Cystinosis

Hypophosphatasia (HPP) is caused by genetic alterations of the ALPL gene, encoding the tissue-nonspecific isozyme of alkaline phosphatase (TNSALP). Here, the purpose was to perform clinical and molecular investigation in a 36-year-old Caucasian woman suspected to present adult HPP. Medical and dental histories were obtained for the proposita and family members, including biochemical, radiographic, and dental assessments. ALPL mutational analysis was performed by the Sanger sequencing method, and the functional impact prediction of the identified mutations was assessed by bioinformatic methods. We identified a novel heterozygous nonsense mutation in the ALPL gene (NM_000478.6:c.768G>A; W[TGG]>*[TGA]) associated with spontaneous vertebral fracture, severe back pain, musculoskeletal pain, low bone density, and short-rooted permanent teeth loss. Functional prediction analysis revealed that the Trp256Ter mutation led to a complete loss of TNSALP crown domain and extensive loss of other functional domains (calcium-binding domain, active site vicinity, and zinc-binding site) and over 60% loss of homodimer interface residues, suggesting that the mutant TNSALP molecules are nonfunctional and form unstable homodimers. Genotyping of the ALPL in the proposita's parents, sister, and niece revealed that in this case, HPP occurred due to a de novo mutation. The present study describes a novel genotype-phenotype and structure-function relationship for HPP, contributing to a better molecular comprehension of HPP etiology and pathophysiology.

Post-transplant G-CSF impedes engraftment of gene-edited human hematopoietic stem cells by exacerbating p53-mediated DNA damage response.

Tracking clonal dynamics, tolerance, and adverse events after CD45-ADC–Conditioned autologous HSPC transplantation in rhesus macaques

Hypophosphatasia (HPP) is a rare, inherited, metabolic disease caused by low activity of tissue non-specific alkaline phosphatase (TNSALP). HPP in adults has a heterogeneous clinical presentation, often with multiple musculoskeletal and systemic manifestations, which can lead to misdiagnosis and substantial delays in diagnosis. To better understand the most common clinical profile of adults with HPP, data from 270 adults with confirmed HPP diagnosis (≥18 year of age, low ALP and/or ALPL mutation) in the Global HPP Registry were analyzed. Most were women (75.2%) with a median age at enrollment into the registry of 50 years and a median ALP activity of 25 U/L (normal range: 40-150 U/L). Based on medical history, pain (74.8%), mostly described as chronic bone pain or generalized body pain, was the leading symptom reported. Dental manifestations, such as early loss of primary teeth, were reported in 60.4% of adults. Skeletal manifestations, such as recurrent and poorly healing fractures or pseudofractures, were observed in 47.8% of adults. A substantial proportion also reported fatigue (35.2%) and muscle weakness (26.3%). Of the adults who had quality of life data available, median (range) of SF-36v2 Physical Component Score was 42.4 (17.9, 63.3; n=203; population norm=50) and pain interference on the BPI-SF was 3.5 (0, 9.5; n=196; 0=does not interfere; 10=completely interferes). Most adults with available data (n=212) reported some disability on the HAQ-DI (66%). Of these 270 adults, 77 were treated with enzyme replacement therapy (asfotase alfa) while 193 had never received treatment. Demographics and baseline ALP activity were similar between ever and never treated adults. Treated patients had a higher occurrence of pain, dental issues, recurrent and poorly healing fractures or pseudofractures, fatigue, and muscle weakness compared with untreated patients. Generally, treated adults reported poorer quality of life before treatment initiation compared with untreated adults, though data were limited in treated adults. Of the patients treated, 61.0% had pediatric-onset HPP which likely reflects the indication for asfotase alfa in most countries; 29.9% had unknown onset. 57.5% of untreated adults had adult-onset HPP, 24.3% had pediatric-onset, and 18.1% has unknown onset. Pain, dental issues, fatigue, recurrent and poorly healing fractures or pseudofractures, and muscle weakness were the most frequently reported symptoms in both adults with pediatric- or adult-onset HPP. These results establish real-world clinical profiles of adults with HPP, both untreated and before treatment start. Similarities in clinical profiles between adults with pediatric-onset and adult-onset HPP suggest that the age at onset itself is of limited clinical utility; current clinical status and the degree of disability are likely more meaningful in making treatment decisions.

3029 – COMBINATORIAL GENE TARGETING IN PRIMARY HUMAN HEMATOPOIETIC STEM AND PROGENITOR CELLS

Capturing Endothelial-to-Hematopoietic Transition Using a Highly Efficient in Vitro Platform

Hypophosphatasia is a rare inborn error of metabolism resulting from mutations in the gene for the tissue-nonspecific isozyme of alkaline phosphatase (TNSALP). There is deficiency of alkaline phosphatase activity leading...

Mir-99 Is a Regulator of Hematopoietic and Leukemic Stem Cell Differentiation