Antisense oligonucleotide (AON)-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy (DMD) patients to restore dystrophin expression by reframing the disrupted... Show moreAntisense oligonucleotide (AON)-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy (DMD) patients to restore dystrophin expression by reframing the disrupted open reading frame of the DMD transcript. However, the treatment efficacy of the already conditionally approved AONs remains low. Aiming to optimize AON efficiency, we assessed exon 53 skipping of the DMD transcript with different chemically modified AONs, all with a phosphorothioate backbone: 2 '-O-methyl (2 ' OMe), locked nucleic acid (LNA)-2 ' OMe, 2 '-fluoro (FRNA), LNA-FRNA, alpha LNA-FRNA, and FANA-LNA-FRNA. Efficient exon 53 skipping was observed with the FRNA, LNA-FRNA, and LNA-2 ' OMe AONs in human control myoblast cultures. Weekly subcutaneous injections (50 mg/kg AON) for a duration of 6 weeks were well tolerated by hDMDdel52/mdx males. Treatment with the LNA-FRNA and LNA-2 ' OMe AONs resulted in pronounced exon 53 skip levels in skeletal muscles and heart up to 90%, but no dystrophin restoration was observed. This discrepancy was mainly ascribed to the strong binding nature of LNA modifications to RNA, thereby interfering with the amplification of the unskipped product resulting in artificial overamplification of the exon 53 skip product. Our study highlights that treatment effect on RNA and protein level should both be considered when assessing AON efficiency. Show less
Engelbeen, S.; Pasteuning-Vuhman, S.; Meulen, J.B. van der; Parmar, R.; Charisse, K.; Sepp-Lorenzino, L.; ... ; Putten, M. van 2022
Downregulation of genes involved in the secondary pathology of Duchenne muscular dystrophy, for example, inflammation, fibrosis, and adiposis, is an interesting approach to ameliorate degeneration... Show moreDownregulation of genes involved in the secondary pathology of Duchenne muscular dystrophy, for example, inflammation, fibrosis, and adiposis, is an interesting approach to ameliorate degeneration of muscle and replacement by fibrotic and adiposis tissue. Small interfering RNAs (siRNAs) are able to downregulate target genes, however, delivery of siRNAs to skeletal muscle still remains a challenge. We investigated delivery of fully chemically modified, cholesterol-conjugated siRNAs targeting Alk4, a nontherapeutic target that is expressed highly in muscle. We observed that a single intravenous or intraperitoneal (IP) injection of 10 mg/kg resulted in significant downregulation of Alk4 mRNA expression in skeletal muscles in both wild-type and mdx mice. Treatment with multiple IP injections of 10 mg/kg led to an overall reduction of Alk4 expression, reaching significance in tibialis anterior (39.7% +/- 6.2%), diaphragm (32.7% +/- 5.8%), and liver (41.3% +/- 29.9%) in mdx mice. Doubling of the siRNA dose did not further increase mRNA silencing in muscles of mdx mice. The chemically modified conjugated siRNAs used in this study are very promising for delivery to both nondystrophic and dystrophic muscles and could have major implications for treatment of muscular dystrophy pathology. Show less
The expression of free fatty acid 1 receptors (FFA1R), activated by long chain fatty acids in human pancreatic beta-cells and enhancing glucose-stimulated insulin secretion are an attractive target... Show moreThe expression of free fatty acid 1 receptors (FFA1R), activated by long chain fatty acids in human pancreatic beta-cells and enhancing glucose-stimulated insulin secretion are an attractive target to treat type 2 diabetes. Yet several clinical studies with synthetic FFA1R agonists had to be discontinued due to cytotoxicity and/or so-called "liver concerns". It is not clear whether these obstructions are FFA1R dependent. In this context we used CHO-AEQ cells expressing the bioluminescent calcium-sensitive protein aequorin to investigate calcium signaling elicited by FFA1 receptor ligands alpha-linolenic acid (ALA), oleic acid (OLA) and myristic acid (MYA). This study revealed complex modulation of intracellular calcium signaling by these fatty acids. First these compounds elicited a typical transient increase of intracellular calcium via binding to FFA1 receptors. Secondly slightly higher concentrations of ALA substantially reduced ATP mediated calcium responses in CHO-AEQ cells and Angiotensin II responses in CHO-AEQ cells expressing human AT 1 receptors. This effect was less pronounced with MYA and OLA and was not linked to FFA1 receptor activation nor to acute cytotoxicity as a result of plasma membrane perturbation. Yet it can be hypothesized that, in line with previous studies, unsaturated long chain fatty acids such as ALA and OLA are capable of inactivating the G-proteins involved in purinergic and Angiotensin AT(1) receptor calcium signaling. Alternatively the ability of fatty acids to deplete intracellular calcium stores might underly the observed cross-inhibition of these receptor responses in the same cells. Show less
Alqallaf, A.; Engelbeen, S.; Palo, A.; Cutrupi, F.; Tanganyika-de Winter, C.; Plomp, J.; ... ; Putten, M. van 2022
Limb girdle muscular dystrophy type 2D (LGMD2D) is characterized by progressive weakening of muscles in the hip and shoulder girdles. It is caused by a mutation in the a-sarcoglycan gene and... Show moreLimb girdle muscular dystrophy type 2D (LGMD2D) is characterized by progressive weakening of muscles in the hip and shoulder girdles. It is caused by a mutation in the a-sarcoglycan gene and results in absence of a-sarcoglycan in the dystrophin-glycoprotein complex. The activin type IIB receptor is involved in the activin/myostatin pathway, with myostatin being a negative regulator of muscle growth. In this study, we investigated the effects of sequestering myostatin by a soluble activin type IIB receptor (sActRIIB) on muscle growth in Sgca-null mice, modelling LGMD2D. Treatment was initiated at 3 weeks of age, prior to the disease onset, or at 9 weeks of age when already in an advanced stage of the disease. We found that early sActRIIB treatment resulted in increased muscle size. However, this led to more rapid decline of muscle function than in saline-treated Sgca-null mice. Furthermore, no histopathological improvements were seen after sActRIIB treatment. When initiated at 9 weeks of age, sActRIIB treatment resulted in increased muscle mass too, but to a lesser extent. No effect of the treatment was observed on muscle function or histopathology. These data show that sActRIIB treatment as a stand-alone therapy does not improve muscle function or histopathology in Sgca-null mice. (c) 2022 Elsevier B.V. All rights reserved. Show less
Engelbeen, S.; Aartsma-Rus, A.; Koopmans, B.; Loos, M.; Putten, M. van 2021
Duchenne muscular dystrophy (DMD) is a severe, progressive neuromuscular disorder caused by mutations in the DMD gene resulting in loss of functional dystrophin protein. The muscle dystrophin... Show moreDuchenne muscular dystrophy (DMD) is a severe, progressive neuromuscular disorder caused by mutations in the DMD gene resulting in loss of functional dystrophin protein. The muscle dystrophin isoform is essential to protect muscles from contraction-induced damage. However, most dystrophin isoforms are expressed in the brain. In addition to progressive muscle weakness, many DMD patients therefore also exhibit intellectual and behavioral abnormalities. The most commonly used mouse model for DMD, the mdx mouse, lacks only the full-length dystrophin isoforms and has been extensively characterized for muscle pathology. In this study, we assessed behavioral effects of a lack of full-length dystrophins on spontaneous behavior, discrimination and reversal learning, anxiety, and short-term spatial memory and compared performance between male and female mdx mice. In contrast to our previous study using only female mdx mice, we could not reproduce the earlier observed reversal learning deficit. However, we did notice small differences in the number of visits made during the Y-maze and dark-light box. Results indicate that it is advisable to establish standard operating procedures specific to behavioral testing in mdx mice to allow the detection of the subtle phenotypic differences and to eliminate inter and intra laboratory variance. Show less
