Non-obese diabetic mice aged 30 to 60 days were treated orally with Cyclosporin at doses of 25, 15 and 2.5 mg/kg every 2 days until 160 days of age. Diabetes developed in 12 out of 18 oil-treated mice (67%), with partial to complete Langerhans' islet destruction associated with lymphocytic infiltration. The non-obese diabetic mice showed a plasma glucose concentration of 6.62 +/- 0.92 mmol/l (mean +/- SD) at 50 days of age. The plasma glucose level of oil-treated non-obese diabetic mice gradually increased after 130 days of age and reached 14.0 to 19.0 mmol/l at 160 days of age, while Cyclosporin-treated non-obese diabetic mice showed neither clear increase of plasma glucose levels nor development of insulitis. The cumulative incidence of diabetes in Cyclosporin-treated mice was significantly lower than that in oil-treated mice (p less than 0.01). Subsequently, Cyclosporin treatment was started after development of glucose intolerance. Twenty-five mg/kg of Cyclosporin was administered every 2 days for 35 days. Cyclosporin appeared to have little therapeutic effect on diabetes in non-obese diabetic mice.

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 would be a useful model to further our understanding of the mcEDS pathophysiology and in the development of novel treatment strategies.

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-D 10); 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-D 10 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-D 10). The vector was injected into both quadriceps femoris muscles of newborn HPP mice at a dose of 4.5 × 10 12 vector genome (v.g.)/body, resulting in 20 U/mL of serum ALP activity. The 4.5 × 10 12 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-D 10 therapy is a promising option for improving the quality of life (QOL) of patients with the infantile form of HPP. Keywords Hypophosphatasia • Adeno-associated virus vector • Enzyme replacement therapy • Alkaline phosphatase • Bone structure Abbreviations AAV Adeno-associated virus ALP Alkaline phosphatase CBA Chicken beta-actin EGFP Enhanced green fluorescence protein Electronic supplementary material The online version of this article (