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.

A new mouse model of Ehlers-Danlos syndrome generated using CRISPR/Cas9-mediated genomic editing.

Musculocontractural Ehlers-Danlos syndrome (mcEDS) is caused by generalized depletion of dermatan sulfate (DS) due to biallelic pathogenic variants in CHST14 encoding dermatan 4-O-sulfotransferase 1 (D4ST1) (mcEDS-CHST14). Here, we generated mouse models for mcEDS-CHST14 carrying homozygous mutations (1 bp deletion or 6 bp insertion/10 bp deletion) in Chst14 through CRISPR/Cas9 genome engineering to overcome perinatal lethality in conventional Chst14-deleted knockout mice. DS depletion was detected in the skeletal muscle of these genome-edited mutant mice, consistent with loss of D4ST1 activity. The mutant mice showed common pathophysiological features, regardless of the variant, including growth impairment and skin fragility. Notably, we identified myopathy-related phenotypes. Muscle histopathology showed variation in fiber size and spread of the muscle interstitium. Decorin localized diffusely in the spread endomysium and perimysium of skeletal muscle, unlike in wild-type mice. The mutant mice showed lower grip strength and decreased exercise capacity compared to wild type, and morphometric evaluation demonstrated thoracic kyphosis in mutant mice. The established CRISPR/Cas9-engineered Chst14 mutant mice could be a useful model to further our understanding of mcEDS pathophysiology and aid in the development of novel treatment strategies.

Myopathy Associated With Dermatan Sulfate-Deficient Decorin and Myostatin in Musculocontractural Ehlers-Danlos Syndrome: A Mouse Model Investigation.

Carbohydrate sulfotransferase 14 (CHST14) encodes dermatan 4-O-sulfotransferase 1, a critical enzyme for dermatan sulfate (DS) biosynthesis. Musculocontractural Ehlers-Danlos syndrome (mcEDS) is associated with biallelic pathogenic variants of CHST14 and is characterized by malformations and manifestations related to progressive connective tissue fragility. We identified myopathy phenotypes in Chst14-deficient mice using an mcEDS model. Decorin is a proteoglycan harboring a single glycosaminoglycan chain containing mainly DS, which are replaced with chondroitin sulfate (CS) in mcEDS patients with CHST14 deficiency. We studied the function of decorin in the skeletal muscle of Chst14-deficient mice because decorin is important for collagen-fibril assembly and has a myokine role in promoting muscle growth. Although decorin was present in the muscle perimysium of wild-type (Chst14+/+ ) mice, decorin was distributed in the muscle perimysium as well as in the endomysium of Chst14-/- mice. Chst14-/- mice had small muscle fibers within the spread interstitium; however, histopathological findings indicated milder myopathy in Chst14-/- mice. Myostatin, a negative regulator of protein synthesis in the muscle, was upregulated in Chst14-/- mice. In the muscle of Chst14-/- mice, decorin was downregulated compared to that in Chst14+/+ mice. Chst14-/- mice showed altered cytokine/chemokine balance and increased fibrosis, suggesting low myogenic activity in DS-deficient muscle. Therefore, DS deficiency in mcEDS causes pathological localization and functional abnormalities of decorin, which causes disturbances in skeletal muscle myogenesis.

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.

High-Level Expression of Alkaline Phosphatase by Adeno-Associated Virus Vector Ameliorates Pathological Bone Structure in a Hypophosphatasia Mouse Model.

Hypophosphatasia (HPP) is a systemic skeletal disease caused by mutations in the gene encoding tissue-nonspecific alkaline phosphatase (TNALP). We recently reported that survival of HPP model mice can be prolonged using an adeno-associated virus (AAV) vector expressing bone-targeted TNALP with deca-aspartate at the C terminus (TNALP-D10); however, abnormal bone structure and hypomineralization remained in the treated mice. Here, to develop a more effective and clinically applicable approach, we assessed whether transfection with TNALP-D10 expressing virus vector at a higher dose than previously used would ameliorate bone structure defects. We constructed a self-complementary AAV8 vector expressing TNALP driven by the chicken beta-actin (CBA) promoter (scAAV8-CB-TNALP-D10). The vector was injected into both quadriceps femoris muscles of newborn HPP mice at a dose of 4.5 × 1012 vector genome (v.g.)/body, resulting in 20 U/mL of serum ALP activity. The 4.5 × 1012 v.g./body-treated HPP mice grew normally and displayed improved bone structure at the knee joints in X-ray images. Micro-CT analysis showed normal trabecular bone structure and mineralization. The mechanical properties of the femur were also recovered. Histological analysis of the femurs demonstrated that ALP replacement levels were sufficient to promote normal, growth plate cartilage arrangement. These results suggest that AAV vector-mediated high-dose TNALP-D10 therapy is a promising option for improving the quality of life (QOL) of patients with the infantile form of HPP.

Tissue-nonspecific alkaline phosphatase promotes the osteogenic differentiation of osteoprogenitor cells.

Tissue-nonspecific alkaline phosphatase (TNAP) is expressed in the calcification sites of the skeletal tissue. It promotes hydroxyapatite crystal formation by degrading inorganic pyrophosphate (PPi) and increasing inorganic phosphate (Pi) concentration. However, abnormalities in Alpl-/- mouse-derived osteoblasts are poorly understood, and the involvement of TNAP in osteoblast differentiation remains unclear. Therefore, in this study, we aimed to investigate the precise role of TNAP in osteoblast differentiation. TNAP inhibition by levamisole, a reversible TNAP inhibitor, suppressed the expression of osteoblast differentiation marker genes in wild-type osteoblastic cells. Alpl overexpression increased the expression of master osteoblast transcription factor genes runt-related transcription factor 2 (Runx2) and Sp7 and the mature osteoblast and osteocyte marker genes, bone γ-carboxyglutamate protein 2 (Bglap2) and dentin matrix protein 1 (Dmp1), respectively in Alpl-deficient osteoblastic cells. TNAP regulated Runx2 expression, which in turn regulated the expression of all other osteoblast markers, except Dmp1. Dmp1 expression was independent of RUNX2 but was dependent on extracellular Pi concentration in Runx2-deficient osteogenic cells. These results suggest that TNAP functions as an osteogenic differentiation regulator either by regulating Runx2 expression or by controlling extracellular Pi concentration.

Method for diagnosing neoplastic lesions by quantitative fluorescence value.

Fluorescence visualization devices (FVs) are useful for detecting malignant lesions because of their simple and noninvasive application. However, their quantitative application has been challenging. This study aimed to quantitatively and statistically evaluate the change in fluorescence intensity (FI) during the progression from normal epithelium to squamous cell carcinoma using a reproducible animal tongue carcinogenesis model. To establish this model, rats were treated with 50 ppm 4-Nitroquinoline 1-oxide (4NQO) in their drinking water for 10, 15, and 20 weeks. After 4NQO administration, each rat tongue was evaluated by gross observation, histology, and FI measurements. Fluorescence images were captured by FV, and ImageJ was used to measure FI, which was analyzed quantitatively and statistically. The establishment of a reproducible tumor progression model was confirmed, showing precancerous lesions (low-grade dysplasia [LGD]), early cancers (high-grade dysplasia/carcinoma in situ [HGD/CIS]), and advanced cancers (Cancer). This carcinogenesis model was quantitatively evaluated by FI. The FI of LGD stage was 54.6, which was highest intensity of all groups. Subsequently, the HGD/CIS and Cancer stages showed decreased FI (HGD/CIS: 46.1, Cancer: 49.1) and manifested as dark spots. This result indicates that FI had more variation and a wider range with increasing tumor progression. We demonstrated that FI migration and an uneven distribution are consistent with tumor progression. Since each step of tumor progression occurs reproducibly in this animal model, statistical evaluation was possible. In addition, tumor progression can be monitored by this new FI analysis method in humans.

Transplantation of human dental pulp stem cells ameliorates brain damage following acute cerebral ischemia.

AIMS: Numerous experimental studies have shown that cellular therapy, including human dental pulp stem cells (DPSCs), is an attractive strategy for ischemic brain injury. Herein, we examined the effects of intravenous DPSC administration after transient middle cerebral artery occlusion in rats. METHODS: Male Sprague-Dawley rats received a transient 90 min middle cerebral artery occlusion. DPSCs (1 × 106 cells) or vehicle were administered via the femoral vein at 0 h or 3 h after ischemia-reperfusion. PKH26, a red fluorescent cell linker, was used to track the transplanted cells in the brain. Infarct volume, neurological deficits, and immunological analyses were performed at 24 h and 72 h after reperfusion. RESULTS: PKH26-positive cells were observed more frequently in the ipsilateral than the contralateral hemisphere. DPSCs transplanted at 0 h after reperfusion significantly reduced infarct volume and reversed motor deficits at 24 h and 72 h recovery. DPSCs transplanted at 3 h after reperfusion also significantly reduced infarct volume and improved motor function compared with vehicle groups at 24 h and 72 h recovery. Further, DPSC transplantation significantly inhibited microglial activation and pro-inflammatory cytokine expression compared with controls at 72 h after reperfusion. Moreover, DPSCs attenuated neuronal degeneration in the cortical ischemic boundary area. CONCLUSIONS: Systemic delivery of human DPSCs after reperfusion reduced ischemic damage and improved functional recovery in a rodent ischemia model, with a clinically relevant therapeutic window. The neuroprotective action of DPSCs may relate to the modulation of neuroinflammation during the acute phase of stroke.

Bone-Targeted Alkaline Phosphatase Treatment of Mandibular Bone and Teeth in Lethal Hypophosphatasia via an scAAV8 Vector.

Hypophosphatasia is an inherited disease caused by mutations in the gene encoding tissue-nonspecific alkaline phosphatase (TNALP), the major symptom of which is hypomineralization of the bones and teeth. We had recently demonstrated that TNALP-deficient (Akp2-/- ) mice, which mimic the phenotype of the severe infantile form of hypophosphatasia, can be treated by intramuscular injection of a self-complementary (sc) type 8 recombinant adeno-associated virus (rAAV8) vector expressing bone-targeted TNALP with deca-aspartates at the C terminus (TNALP-D10) via the muscle creatine kinase (MCK) promoter. In this study, we focused on the efficacy of this scAAV8-MCK-TNALP-D10 treatment on the mandibular bone and teeth in neonatal Akp2-/- mice. Upon scAAV8-MCK-TNALP-D10 injection, an improvement of mandibular growth was observed by X-ray analysis. Micro-computed tomography analysis revealed progressive mineralization of the molar root in the treated Akp2-/- mice, and morphometric parameters of the alveolar bone were improved. These results suggest that the mandibular bones and teeth of hypophosphatasia were effectively treated by muscle directed rAAV-mediated TNALP-D10 transduction. Our strategy would be promising for future hypophosphatasia gene therapy because it induces dentoalveolar mineralization and reduces the risk of tooth exfoliation.

Impact of Dental Pulp Stem Cells Overexpressing Hepatocyte Growth Factor after Cerebral Ischemia/Reperfusion in Rats.

Hepatocyte growth factor (HGF) has neuroprotective effects against ischemia-induced injuries. Dental pulp stem cell (DPSC) transplantation attenuates tissue injury in the brain of rats with post-transient middle cerebral artery occlusion. We sought to determine whether DPSCs that overexpress HGF can enhance their therapeutic effects on brain damage post-ischemia/reperfusion injury. Treatment with DPSCs overexpressing HGF reduced infarct volumes compared to unmodified DPSC treatment at 3 and 7 days post-transient middle cerebral artery occlusion. The use of unmodified DPSCs and DPSCs overexpressing HGF was associated with improved motor function compared to that with administration of vehicle at 7 days post-transient middle cerebral artery occlusion. DPSCs overexpressing HGF significantly inhibited microglial activation and pro-inflammatory cytokine production along with suppression of neuronal degeneration. Post-reperfusion, DPSCs overexpressing HGF attenuated the decreases in tight junction proteins, maintained blood-brain barrier integrity, and increased microvessel density in peri-infarct areas. The administration of DPSCs overexpressing HGF during the acute phase of stroke increased their neuroprotective effects by modulating inflammation and blood-brain barrier permeability, thereby promoting improvements in post-ischemia/reperfusion brain injury.

Mesenchymal Stem Cells Overexpressing Interleukin-10 Promote Neuroprotection in Experimental Acute Ischemic Stroke.

Interleukin (IL)-10 is a contributing factor to neuroprotection of mesenchymal stem cell (MSC) transplantation after ischemic stroke. Our aim was to increase therapeutic effects by combining MSCs and ex vivo IL-10 gene transfer with an adeno-associated virus (AAV) vector using a rat transient middle cerebral artery occlusion (MCAO) model. Sprague-Dawley rats underwent 90 min MCAO followed by intravenous administration of MSCs alone or IL-10 gene-transferred MSCs (MSC/IL-10) at 0 or 3 hr after ischemia reperfusion. Infarct lesions, neurological deficits, and immunological analyses were performed within 7 days after MCAO. 0-hr transplantation of MSCs alone and MSC/IL-10 significantly reduced infarct volumes and improved motor function. Conversely, 3-hr transplantation of MSC/IL-10, but not MSCs alone, significantly reduced infarct volumes (p < 0.01) and improved motor function (p < 0.01) compared with vehicle groups at 72 hr and 7 days after MCAO. Immunological analysis showed that MSC/IL-10 transplantation significantly inhibits microglial activation and pro-inflammatory cytokine expression compared with MSCs alone. Moreover, overexpressing IL-10 suppressed neuronal degeneration and improved survival of engrafted MSCs in the ischemic hemisphere. These results suggest that overexpressing IL-10 enhances the neuroprotective effects of MSC transplantation by anti-inflammatory modulation and thereby supports neuronal survival during the acute ischemic phase.

Treatment of hypophosphatasia by muscle-directed expression of bone-targeted alkaline phosphatase via self-complementary AAV8 vector.

Hypophosphatasia (HPP) is an inherited disease caused by genetic mutations in the gene encoding tissue-nonspecific alkaline phosphatase (TNALP). This results in defects in bone and tooth mineralization. We recently demonstrated that TNALP-deficient (Akp2 (-/-) ) mice, which mimic the phenotype of the severe infantile form of HPP, can be treated by intravenous injection of a recombinant adeno-associated virus (rAAV) expressing bone-targeted TNALP with deca-aspartates at the C-terminus (TNALP-D10) driven by the tissue-nonspecific CAG promoter. To develop a safer and more clinically applicable transduction strategy for HPP gene therapy, we constructed a self-complementary type 8 AAV (scAAV8) vector that expresses TNALP-D10 via the muscle creatine kinase (MCK) promoter (scAAV8-MCK-TNALP-D10) and examined the efficacy of muscle-directed gene therapy. When scAAV8-MCK-TNALP-D10 was injected into the bilateral quadriceps of neonatal Akp2 (-/-) mice, the treated mice grew well and survived for more than 3 months, with a healthy appearance and normal locomotion. Improved bone architecture, but limited elongation of the long bone, was demonstrated on X-ray images. Micro-CT analysis showed hypomineralization and abnormal architecture of the trabecular bone in the epiphysis. These results suggest that rAAV-mediated, muscle-specific expression of TNALP-D10 represents a safe and practical option to treat the severe infantile form of HPP.

Prevention of Lethal Murine Hypophosphatasia by Neonatal Ex Vivo Gene Therapy Using Lentivirally Transduced Bone Marrow Cells.

Hypophosphatasia (HPP) is an inherited skeletal and dental disease caused by loss-of-function mutations in the gene that encodes tissue-nonspecific alkaline phosphatase (TNALP). The major symptoms of severe forms of the disease are bone defects, respiratory insufficiency, and epileptic seizures. In 2015, enzyme replacement therapy (ERT) using recombinant bone-targeted TNALP with deca-aspartate (D10) motif was approved to treat pediatric HPP patients in Japan, Canada, and Europe. However, the ERT requires repeated subcutaneous administration of the enzyme because of the short half-life in serum. In the present study, we evaluated the feasibility of neonatal ex vivo gene therapy in TNALP knockout (Akp2(-/-)) HPP mice using lentivirally transduced bone marrow cells (BMC) expressing bone-targeted TNALP in which a D10 sequence was linked to the C-terminus of soluble TNALP (TNALP-D10). The Akp2(-/-) mice usually die within 20 days because of growth failure, epileptic seizures, and hypomineralization. However, an intravenous transplantation of BMC expressing TNALP-D10 (ALP-BMC) into neonatal Akp2(-/-) mice prolonged survival of the mice with improved bone mineralization compared with untransduced BMC-transplanted Akp2(-/-) mice. The treated Akp2(-/-) mice were normal in appearance and experienced no seizures during the experimental period. The lentivirally transduced BMC were efficiently engrafted in the recipient mice and supplied TNALP-D10 continuously at a therapeutic level for at least 3 months. Moreover, TNALP-D10 overexpression did not affect multilineage reconstitution in the recipient mice. The plasma ALP activity was sustained at high levels in the treated mice, and tissue ALP activity was selectively detected on bone surfaces, not in the kidneys or other organs. No ectopic calcification was observed in the ALP-BMC-treated mice. These results indicate that lentivirally transduced BMC can serve as a reservoir for stem cell-based ERT to rescue the Akp2(-/-) phenotype. Neonatal ex vivo gene therapy thus appears to be a possible treatment option for treating severe HPP.