作者: Bennett, C Frank ; Wirth, Brunhilde ; Kononenko, Natalia L ; Upadhyay, Aaradhita ; Schneider, Svenja ; Ling, Karen K ; Rigo, Frank ; Rombo, Roman ; Torres-Benito, Laura ; Grysko, Vanessa

Spinal muscular atrophy (SMA) is a neuromuscular disease causing the most frequent genetic childhood lethality. Recently, nusinersen, an antisense oligonucleotide (ASO) that corrects SMN2 splicing and thereby increases full-length SMN protein, has been approved by the FDA and EMA for SMA therapy. However, the administration of nusinersen in severe and/or post-symptomatic SMA-affected individuals is insufficient to counteract the disease. Therefore, additional SMN-independent therapies are needed to support the function of motoneurons and neuromuscular junctions. We recently identified asymptomatic SMN1-deleted individuals who were protected against SMA by reduced expression of neurocalcin delta (NCALD). NCALD reduction is proven to be a protective modifier of SMA across species, including worm, zebrafish, and mice. Here, we identified Ncald-ASO3-out of 450 developed Ncald ASOs-as the most efficient and non-toxic ASO for the CNS, by applying a stepwise screening strategy in cortical neurons and adult and neonatal mice. In a randomized-blinded preclinical study, a single subcutaneous low-dose SMN-ASO and a single intracerebroventricular Ncald-ASO3 or control-ASO injection were presymptomatically administered in a severe SMA mouse model. NCALD reduction of >70% persisted for about 1 month. While low-dose SMN-ASO rescues multiorgan impairment, additional NCALD reduction significantly ameliorated SMA pathology including electrophysiological and histological properties of neuromuscular junctions and muscle at P21 and motoric deficits at 3 months. The present study shows the additional benefit of a combinatorial SMN-dependent and SMN-independent ASO-based therapy for SMA. This work illustrates how a modifying gene, identified in some asymptomatic individuals, helps to develop a therapy for all SMA-affected individuals.

2019-07-01·European review for medical and pharmacological sciences

MiR-140-3p overexpression activates the Wnt signaling pathway to promote fracture healing.

Article

作者: Liu, Q-P ; Chen, L ; Zheng, H ; Lin, Z ; Wu, T-H ; Chen, S-J ; Chen, Y-H

OBJECTIVETo study the mechanism of micro ribonucleic acid (miR)-140-3p participating in the regulation of fracture healing in rats.MATERIALS AND METHODSA total of 50 male Sprague-Dawley (SD) rats were randomly divided into five groups, namely, group A [phosphate-buffered saline (PBS)] (n=10), group B (miR-140-3p mimics) (n=10), group C [ mimics negative control (NC)] (n=10), group D [antisense oligonucleotide (ASO)-miR-140-3p] (n=10), and group E (ASO NC) (n=10). A rat model of fracture was established on all the rats through the operation. From the successful establishment of the model, the rats in group A were intraperitoneally injected with 50 μL PBS (2 nmol) once a week for 6 weeks, and those in group B, C, D, and E were injected with equivalent volume of miR-140-3p mimics, mimics NC, ASO-miR-140-3p, and ASO NC, respectively, once a week since the successful establishment of model for 6 weeks. The fracture healing in the rats was evaluated via imaging. Meanwhile, Real Time-Polymerase Chain Reaction (RT-PCR) was applied to detect the expression of miR-140-3p in the five groups. Wnt and β-catenin expressions in the five groups were detected by means of Western blotting (WB). Alkaline phosphatase (ALP) and its quantized statistical value in the five groups were detected through immunohistochemical staining.RESULTSThe expression of miR-140-3p was stimulated in miR-140-3p mimics group and inhibited in ASO-miR-140-3p group. The detection of the miR-140-3p expression level in the five groups via RT-PCR showed that miR-140-3p mimics group had a remarkably higher miR-140-3p expression than the other four groups. The differences were statistically significant (p<0.05). The WB assay verified that the Wnt and β-catenin expressions in miR-140-3p mimics group were notably higher than those in control groups, and there were statistically significant differences (p<0.05). Compared with those in the groups injected with PBS, ASO miR-140-3p, mimics NC, and ASO NC, there were evidently more callus tissues, better healed and more blurred fracture lines, as well as no translocation and looseness of internal fixation, in the group injected with miR-140-3p mimics, suggesting that the stimulation of the miR-140-3p expression promotes the fracture healing in the rats. The results of immunohistochemical staining indicated that the number of ALP-positive osteoblasts in the rats in miR-140-3p mimics group was increased markedly in comparison with that in the remaining groups (p<0.05), implying that the differentiation of osteoblasts in the rats was affected in miR-140-3p mimics group.CONCLUSIONSThe overexpressed miR-140-3p in the rats with fracture can promote fracture healing by activating the Wnt signaling pathway.

International Journal of Molecular Sciences

Long-Term SMN- and Ncald-ASO Combinatorial Therapy in SMA Mice and NCALD-ASO Treatment in hiPSC-Derived Motor Neurons Show Protective Effects

Article

作者: Zilio, Eleonora ; Ling, Karen K. ; Bennett, C. Frank ; Wirth, Brunhilde ; Muiños-Bühl, Anixa ; Rigo, Frank ; Rombo, Roman

For SMA patients with only two SMN2 copies, available therapies might be insufficient to counteract lifelong motor neuron (MN) dysfunction. Therefore, additional SMN-independent compounds, supporting SMN-dependent therapies, might be beneficial. Neurocalcin delta (NCALD) reduction, an SMA protective genetic modifier, ameliorates SMA across species. In a low-dose SMN-ASO-treated severe SMA mouse model, presymptomatic intracerebroventricular (i.c.v.) injection of Ncald-ASO at postnatal day 2 (PND2) significantly ameliorates histological and electrophysiological SMA hallmarks at PND21. However, contrary to SMN-ASOs, Ncald-ASOs show a shorter duration of action limiting a long-term benefit. Here, we investigated the longer-term effect of Ncald-ASOs by additional i.c.v. bolus injection at PND28. Two weeks after injection of 500 µg Ncald-ASO in wild-type mice, NCALD was significantly reduced in the brain and spinal cord and well tolerated. Next, we performed a double-blinded preclinical study combining low-dose SMN-ASO (PND1) with 2× i.c.v. Ncald-ASO or CTRL-ASO (100 µg at PND2, 500 µg at PND28). Ncald-ASO re-injection significantly ameliorated electrophysiological defects and NMJ denervation at 2 months. Moreover, we developed and identified a non-toxic and highly efficient human NCALD-ASO that significantly reduced NCALD in hiPSC-derived MNs. This improved both neuronal activity and growth cone maturation of SMA MNs, emphasizing the additional protective effect of NCALD-ASO treatment.

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适应症	最高研发状态	国家/地区	公司	日期
脊髓性肌萎缩	临床前	德国	University of Cologne Executive School GmbH	2023-02-20
脊髓性肌萎缩	临床前	美国	Ionis Pharmaceuticals, Inc.	2023-02-20