Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by out-of-frame or nonsense mutation in the dystrophin gene. It begins with a loss of ambulation between 9 and 14 years of age, followed by various other symptoms including cardiac dysfunction. Exon skipping of patients’ DMD pre-mRNA induced by antisense oligonucleotides (AOs) is expected to produce shorter but partly functional dystrophin proteins, such as those possessed by patients with the less severe Becker muscular dystrophy. We are working on developing modified nucleotides, such as 2′-O,4′-C-ethylene-bridged nucleic acids (ENAs), possessing high nuclease resistance and high affinity for complementary RNA strands. Here, we demonstrate the preclinical characteristics (exon-skipping activity in vivo, stability in blood, pharmacokinetics, and tissue distribution) of renadirsen, a novel AO modified with 2′-O-methyl RNA/ENA chimera phosphorothioate designed for dystrophin exon 45 skipping and currently under clinical trials. Notably, systemic delivery of renadirsen sodium promoted dystrophin exon skipping in cardiac muscle, skeletal muscle, and diaphragm, compared with AOs with the same sequence as renadirsen but conventionally modified by PMO and 2′OMePS. These findings suggest the promise of renadirsen sodium as a therapeutic agent that improves not only skeletal muscle symptoms but also other symptoms in DMD patients, such as cardiac dysfunction.

We investigated whether neural crest-derived cardiac resident cells contribute to the restoration of intrinsic adrenergic function following transplantation in mice. Transplanted heart shows partial restoration of cardiac adrenergic activity with time. Both the intrinsic cardiac adrenergic system and extrinsic sympathetic re-innervation contribute to neuronal remodelling in the transplanted heart. Little is known about the origin and function of the intrinsic system in the transplanted heart. Heart from the protein 0-Cre/Floxed-Enhanced Green Fluorescent Protein double-transgenic mouse was transplanted onto the abdominal aorta of the non-obese diabetic/severe combined immunodeficient mouse to trace the fate of cardiac resident neural crest-derived cells. Sympathetic nerve fibres, which are predominantly localized to the epicardial surface of the heart, disappeared in the transplanted heart. Intramyocardial neural crest cells increased immediately, while neural crest-derived nucleated tyrosine hydroxylase (TH)-immunoreactive cells increased over 2 weeks following transplantation. The mRNA expression levels of TH, dopamine-β-hydroxylase and phenylethanolamine N-methyltransferase, and the tissue content of catecholamines in the transplanted hearts increased with time in association with an increase in the number of neural crest-derived nucleated TH-immunoreactive cells and tissue nerve growth factor levels. Iodine-123-metaiodobenzylguanidine scintigraphy showed that the uptake ability of transplanted heart for catecholamines also recovered with time. Finally, the chronotropic response to tyramine both in vivo and ex vivo reappeared 2 weeks after transplantation. Neural crest-derived adrenergic cells increased following heart transplantation. The restoration of cardiac sympathetic activities in transplanted heart is tightly coupled with an increase in the number of neural crest-derived adrenergic cells.

Plasma aldosterone concentration increases in proportion to the severity of heart failure, even during treatment with renin-angiotensin system inhibitors. This study investigated alternative regulatory mechanisms of aldosterone production that are significant in heart failure. Dahl salt-sensitive rats on a high-salt diet, a rat model of heart failure with cardio-renal syndrome, had high plasma aldosterone levels and elevated β3-adrenergic receptor expression in hypoxic zona glomerulosa cells. In H295R cells (a human adrenocortical cell line), hypoxia-induced β3-adrenergic receptor expression. Hypoxia-mediated β3-adrenergic receptor expression augmented aldosterone production by facilitating hydrolysis of lipid droplets though ERK-mediated phosphorylation of hormone-sensitive lipase, also known as cholesteryl ester hydrolase. Hypoxia also accelerated the synthesis of cholesterol esters by acyl-CoA:cholesterol acyltransferase, thereby increasing the cholesterol ester content in lipid droplets. Thus, hypoxia enhanced aldosterone production by zona glomerulosa cells via promotion of the accumulation and hydrolysis of cholesterol ester in lipid droplets. In conclusion, hypoxic zona glomerulosa cells with heart failure show enhanced aldosterone production via increased catecholamine responsiveness and activation of cholesterol trafficking, irrespective of the renin-angiotensin system.

Glycogen storage disease type 1a (GSD1a) is caused by a congenital deficiency of glucose-6-phosphatase-α (G6Pase-α, encoded by G6PC), which is primarily associated with life-threatening hypoglycemia. Although strict dietary management substantially improves life expectancy, patients still experience intermittent hypoglycemia and develop hepatic complications. Emerging therapies utilizing new modalities such as adeno-associated virus and mRNA with lipid nanoparticles are under development for GSD1a but potentially require complicated glycemic management throughout life. Here, we present an oligonucleotide-based therapy to produce intact G6Pase-α from a pathogenic human variant, G6PC c.648G>T, the most prevalent variant in East Asia causing aberrant splicing of G6PC. DS-4108b, a splice-switching oligonucleotide, was designed to correct this aberrant splicing, especially in liver. We generated a mouse strain with homozygous knockin of this variant that well reflected the pathophysiology of patients with GSD1a. DS-4108b recovered hepatic G6Pase activity through splicing correction and prevented hypoglycemia and various hepatic abnormalities in the mice. Moreover, DS-4108b had long-lasting efficacy of more than 12 weeks in mice that received a single dose and had favorable pharmacokinetics and tolerability in mice and monkeys. These findings together indicate that this oligonucleotide-based therapy could provide a sustainable and curative therapeutic option under easy disease management for GSD1a patients with G6PC c.648G>T.