Biology subjects

Stan, T. L.

Publications and source records attributed to Stan, T. L..

4 recordsLinked to original sources

bioRxiv2026

Sequence determinants of efficient exon 44 skipping in Duchenne muscular dystrophy define design principles for steric-blocking antisense oligonucleotides

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that disrupt the reading frame and abolish expression of functional dystrophin protein. Antisense oligonucleotides (ASO) can restore production of partially functional dystrophins by inducing exon skipping to restore the reading frame of dystrophin transcripts. While exon skipping is an FDA approved therapeutic strategy, there are currently no approved therapies for patients amenable to exon 44 skipping (8% of DMD patients). Here, we carried out a discovery campaign to identify phosphorothioate (PS) ASOs that efficiently induce exon 44 skipping and to define key sequence and chemistry features associated with activity. A tiling and micro-tiling approach with 18mer fully PS and 2-O-methoxyethyl (2MOE) modified ASOs in patient-derived myotubes identified five exonic target regions that promote skipping. ASO activity was strongly correlated across skeletal muscle and iPSC-derived cardiomyocytes, indicating similar exon 44 splicing regulation across cell types. Optimization studies showed that for 2MOE PS ASOs, 16-20mers were generally most active, while longer ASOs often had reduced activity due in part to impaired productive uptake into cells. Swapping out 2MOE modifications at both terminal positions for locked nucleic acids (LNAs) rarely improved activity and could also reduce it. Finally, promising candidates were tested in a humanized mouse model with an exon 44 skippable deletion, where one ASO outperformed others, inducing dose-dependent exon 44 skipping and dystrophin restoration in mouse models. These findings define practical design rules for exon 44-targeted ASOs and provide a foundation for therapeutic development.

molecular biology↗

bioRxiv2025

Four new Duchenne muscular dystrophy mouse models with clinically relevant exon deletions in the human DMD gene

Mutation specific therapeutic approaches, like exon skipping or gene-editing, hold promise for the treatment of Duchenne muscular dystrophy (DMD). Translatability of preclinical studies investigating these approaches could greatly be improved through the use of humanized mouse models, as these allow preclinical testing of human specific sequences. We developed four novel humanized DMD mouse models with either a deletion of exon 44, 45, 51 or 53 in the human DMD gene, in a mouse dystrophin negative background (mdx mouse; exon 23 nonsense mutation). Our optimized prescreening pipeline allowed us to do so very efficiently with the CRISPR-Cas9 technology. We confirmed either complete lack of dystrophin, or expression of trace levels, which led to development of muscle pathology consisting of muscle fiber de-, and regeneration, inflammation and fibrosis in young adult mice. Intramuscular treatment with vivo-morpholinos targeting a flanking exon induced exon skipping in the DMD strains, which restored the disrupted open reading frame and subsequently dystrophin expression. This validates these models as valuable tools for preclinical studies investigating human sequence specific therapeutic approaches for DMD. Summary statementHumanized Duchenne muscular dystrophy mouse models were created with deletions of exon 44, 45, 51 or 53 in the human DMD gene. These dystrophic models allow preclinical testing of human-specific dystrophin restoring approaches.

neuroscience↗

bioRxiv2024

Investigating the effects of prednisolone on behavior in mouse models of Duchenne muscular dystrophy

BackgroundNext to progressive muscle loss, Duchenne muscular dystrophy patients suffer from behavioral and cognitive problems. This is due to mutations in the DMD gene, that result in the lack of dystrophin in both the muscles and brain. As part of the standards of care, patients receive corticosteroids (prednisolone or deflazacort) to slow down muscle degeneration. The precise consequences of chronic corticosteroid usage on the behavior of DMD patients remain unclear, mainly due to challenges of recruiting corticosteroid naive patients into clinical studies. ObjectiveThis study used DMD mouse models, representing mutations resulting in lack of one or more dystrophin isoforms, to analyze the effects of corticosteroid treatment on different behavioral domains. MethodsPrednisolone (PDN) or placebo was administered via a subcutaneous 60-day slow release pellet (66 {micro}g/day) and mice were subjected to several behavioral tests. ResultsUnfortunately, the pellet only exposed mice to PDN for half of the intended duration. During the time of PDN exposure, we found a small amelioration in anxiety but were unable to find any differences in social interaction and spatial learning and memory. ConclusionsShort term exposure to PDN via a slow release pellet does not seem to negatively affect anxiety, social interaction or spatial learning and memory. We cannot rule out that a longer treatment period than 4 weeks would affect behavior in DMD mice.

animal behavior and cognition↗

bioRxiv2023

The vitamin B3 analogue nicotinamide riboside has only very minor effects on reducing muscle damage in mdx mice

BACKGROUNDVitamin B3 analogue nicotinamide riboside (NR) has been suggested to have beneficial effects on muscle pathology in a mouse model for Duchenne muscular dystrophy (DMD). In muscle dystrophy, NR is thought to act acts by increasing levels of NAD+, to improve mitochondrial functioning and reduce muscle pathology. OBJECTIVEWe here aimed to validate the effects of NR to improve muscle quality after eight weeks of treatment in two different mouse models for DMD: the commonly used mdx mouse on a C57BL/10 background (BL10mdx) and the more severely affected mdx mouse on a DBA/2J background (D2-mdx). METHODSTo study in more detail whether NR treatment had an impact on muscle pathology, we assessed the expression levels of several markers for DMD pathology (fibrosis, regeneration and inflammation) in diaphragm. RESULTSOur data showed a trend for increase in NAD+-levels in blood; only in the D2-mdx NR-treated mice the NAD+-levels were slightly increased. These markers were elevated in mdx models compared to controls, but not affected by the NR treatment. Histological analysis of muscle tissues indicated a mild treatment effect in D2-mdx mice. CONCLUSIONSBased on our results, testing NR treatment in clinical trials in DMD patients is not warranted.

genetics↗