Biology subjects

Lin, K.-H.

Publications and source records attributed to Lin, K.-H..

8 recordsLinked to original sources

bioRxiv2026

In vivo gene disruption and homology-directed repair in muscles and muscle stem cells using CRISPR/Cas9

Programmable endonucleases such as CRISPR/Cas9 provide powerful tools to edit mammalian genomes by engaging cellular mechanisms of DNA double-strand break (DSB) repair. CRISPR-catalysed homology-directed repair (CRISPR-HDR), though generally less efficient than other modes of DNA repair, holds particular promise to enable precise sequence replacement by targeted insertion of a homologous DNA template1,2. While recent studies have reported appreciable levels of HDR in cardiomyocytes in vivo3, skeletal muscle myofibres have historically been considered refractory to HDR-mediated genome editing4. Furthermore, how repair outcomes differ across tissues after systemic delivery of CRISPR/Cas9 editors, whether precise HDR editing can be achieved in regenerative tissue stem cells, and how developmental timing influences accessibility to CRISPR-induced repair remain unclear. Here, we use an adeno-associated virus (AAV)-delivered in vivo GFP-to-BFP colour-switching reporter system (AAV-GFP-to-BFP) to examine in vivo CRISPR-HDR with cellular- and tissue-level resolution. We find that postnatal cardiac muscle, skeletal muscle, and muscle stem cells undergo templated HDR at different rates across discrete developmental stages in mice. While HDR-edited muscle stem cells and myofibres were readily detectable after in vivo editing in juvenile mice, editing in neonatal mice yielded more efficient HDR in cardiac tissue. Based on these results, we adapted the CRISPR-HDR approach to rescue the therapeutically relevant Dmd mutation in mdx mice, demonstrating recoding to the wild-type protein sequence in both skeletal and cardiac muscles. These results provide a framework for advancing donor-templated DNA repair in living postnatal animals, and reveal unexpected cellular, developmental, and disease-related constraints on precise, therapeutic in vivo gene correction.

cell biology↗

bioRxiv2026

In vivo base editing via single myotrophic adeno-associated viruses in dystrophic mouse muscle and satellite cells

Duchenne muscular dystrophy (DMD) is the most common, lethal X-linked neuromuscular disorder of childhood and is caused by mutations in the Dmd gene that disrupt dystrophin expression. Although adeno-associated virus-mediated gene therapies hold tremendous promise for DMD treatment, their clinical applications have been limited by dose-dependent vector and genome-level toxicities. Here, we developed and tested a single-vector adenine base editing strategy as a potentially safer genome editing approach to recode the pathogenic nonsense mutation into a benign missense mutation in mdx4cvDMD mouse model. Delivered using a muscle-tropic adeno-associated virus (MyoAAV) at a clinically-feasible dose (4E13 VG/kg), this strategy enabled detectable molecular recoding of the mdx4cv mutation in mice ranging in age from 3 days to 6 months. Yet, the overall efficiency and therapeutic impact of in vivo base editing with this system was highest in mice treated at the juvenile stage, with animals administered MyoAAV vectors at 3 weeks of age showing robust recovery of dystrophin expression and significant improvement in muscle contractile properties only one month later. Notably, introduction of adenine base editors either earlier in development, in neonatal mice, or later, in adulthood, yielded substantially lower editing efficiencies, particularly in muscle satellite cells whose editing is essential to ensure durable rescue of dystrophin expression in growing and regenerating muscle. Taken together, these results demonstrate the therapeutic potential of single-vector adenine base editing for DMD and underscore the importance of recipient age and disease stage in achieving optimal treatment outcomes for this and other genetic muscle disorders.

cell biology↗

bioRxiv2025

Evolutionarily Conserved Heat-Induced Chromatin Dynamics Drive Heat Stress Responses in Plants

Eukaryotic organisms remodel chromatin landscapes to regulate gene expression in response to environmental stress. In plants, heat stress (HS) induces widespread chromatin changes, yet the role of heat-responsive Heat Shock Transcription Factors (HSFs) in chromatin remodeling and their evolutionary conservation remains unclear. Using chromatin accessibility profiling and transcriptomics in Marchantia polymorpha hsf mutants, we identify HSFA1 as a key determinant in positioning cis-regulatory elements (CREs) for HS-induced gene activation, a mechanism conserved across land plants, mice, and human cells. By integrating gene regulatory network modeling, we identify parallel transcription factor subnetworks, with MpWRKY10 and MpABI5B acting as indirect regulators of HS responses via phenylpropanoid pathways and general stress signaling. We further explore crosstalk between HS and abscisic acid (ABA) signaling, showing that while ABA modulates gene expression in an HSFA1-dependent manner, it does not induce broad chromatin remodeling, positioning it as a downstream regulator rather than a primary determinant of chromatin dynamics. To extend these insights, we develop a cross-species and cross-condition machine learning framework that accurately predicts chromatin accessibility and gene expression, demonstrating a conserved regulatory logic of stress-responsive chromatin and transcription dynamics. Our findings provide a conceptual framework for understanding how TFs coordinate chromatin architecture to drive stress adaptation in plants and potentially other eukaryotes.

plant biology↗

bioRxiv2025

HeteroRepur: Efficient Modeling of Heterogeneous Disease Graph for Depression Drug Repurposing using Heterogeneous Graph Neural Networks

ObjectiveMajor Depressive Disorder (MDD) is one of the most prevalent psychiatric disorders, yet existing treatments often result in suboptimal responses and encounter high recurrence rate. Repositioning existing medications, which often significantly reduces the required time and budget, has been demonstrated to be a promising strategy for drug discovery of multiple central nervous system (CNS) disorders. However, an efficient modeling strategy of heterogeneous semantic information among biological networks has yet to be developed for drug repurposing. Material and MethodsIn this study, we proposed HeteroRepur, by integrating heterogeneous graph neural networks to learn interaction features of Depression Drug Repurposing Graph (DDRG) and then classify drugs. ResultsThe DDRG embeddings learned by HeteroRepur accurately capture graph information, including 89 types of interaction or associations among drugs, genes, pathways, and GOs, as well as disease genetic network of MDD. In drug classification task, HeteroRepur outperforms various baseline models, including existing graph neural network approaches that do not consider heterogeneous network information. Finally, HeteroRepur predicts new candidates for treating MDD, including CNS drugs that have the potential to be extended to MDD, and dietary supplements that might benefit depression patients by elevating essential nutrient levels. ConclusionHeteroRepur outperforms traditional graph-based approaches, and can be used to rank promising drugs among complex disease networks with heterogeneous topology.

bioinformatics↗

bioRxiv2024

Stabilization of AMPK/PFKL/RPIA in the Glycolytic Bodies Transduces IL6/STAT3 Signal in Hepatocarcinogenesis

Metabolic reprogramming is a pivotal characteristic of cancer, yet the intricate interplay between glycolysis and the pentose phosphate pathway (PPP) remains elusive. This study unveils the pivotal role of 6-phosphofructokinase liver type (PFKL) in glycolysis and ribose 5-phosphate isomerase A (RPIA) in PPP, orchestrating liver tumorigenesis. PFKL, the rate-limiting enzyme in glycolysis, stabilizes RPIA by impeding ubiquitination/proteasome activity. The pro-inflammatory and tumor cytokine interleukin 6 activates pSTAT3 which binds to the promoter region and activates AMPK and PFKL transcription. Furthermore, pAMPK stabilizes PFKL protein by preventing proteasome degradation in hepatoma cells. Inhibiting PFKL, AMPK, and STAT3 genetically or pharmacologically can reduce glycolysis, ATP production, resulting in reduction of hepatoma cell proliferation and migration. Intriguingly, the PFKL, AMPK, RPIA, and PKM2 are co-localized in the Glycolytic body (G-body) which starts forming at chronic hepatitis, dramatically increases during active hepatitis, and the size of G-bodies becomes bigger from cirrhosis to hepatocellular carcinoma. Furthermore, using Bimolecular fluorescence complementation (BiFC) assay, we demonstrated that PFKL and RPIA direct interacts. Targeting AMPK or STAT3 significantly reduced tumor formation and lipid accumulation in zebrafish models, suggesting the STAT3/AMPK/PFKL axis as a potential therapeutic avenue for liver cancer treatment.

cancer biology↗

bioRxiv2023

The role of satellite cell-derived TRIM28 in mechanical load- and injury-induced myogenesis

Satellite cells are skeletal muscle stem cells that contribute to postnatal muscle growth, and they endow skeletal muscle with the ability to regenerate after a severe injury. Here we discovered that this myogenic potential of satellite cells requires a protein called tripartite motif-containing 28 (TRIM28). Unexpectedly, multiple lines of both in vitro and in vivo evidence revealed that the myogenic function of TRIM28 is not dependent on changes in the phosphorylation of its serine 473 residue. Moreover, the functions of TRIM28 were not mediated through the regulation of satellite cell proliferation or differentiation. Instead, our findings indicate that TRIM28 regulates the ability of satellite cells to progress through the process of fusion. Specifically, we discovered that TRIM28 controls the expression of a fusogenic protein called myomixer and concomitant fusion pore formation. Collectively, the outcomes of this study expose the framework of a novel regulatory pathway that is essential for myogenesis. TeaserTRIM28 regulates the induction of myomixer expression and fusion pore formation during myogenesis.

cell biology↗

bioRxiv2022

A sequential two-step priming scheme reproduces diversity in synaptic strength and short-term plasticity

Glutamatergic synapses display variable strength and diverse short-term plasticity (STP), even for a given type of connection. Using non-negative tensor factorization (NTF) and conventional state modelling, we demonstrate that a kinetic scheme consisting of two sequential and reversible steps of release-machinery assembly and a final step of synaptic vesicle (SV) fusion reproduces STP and its diversity among synapses. Analyzing transmission at calyx of Held synapses reveals that differences in synaptic strength and STP are not primarily caused by variable fusion probability (pfusion) but determined by the fraction of docked synaptic vesicles equipped with a mature release machinery. Our simulations show, that traditional quantal analysis methods do not necessarily report pfusion of SVs with a mature release machinery but reflect both pfusion and the distribution between mature and immature priming states at rest. Thus, the approach holds promise for a better mechanistic dissection of the roles of presynaptic proteins in the sequence of SV docking, two-step priming and fusion and suggests a mechanism for activity-induced redistribution of synaptic efficacy.

neuroscience↗

bioRxiv2018

Resistance from Afar: Distal Mutation V36M Allosterically Modulates the Active Site to Accentuate Drug Resistance in HCV NS3/4A Protease

Hepatitis C virus rapidly evolves, conferring resistance to direct acting antivirals. While resistance via active site mutations in the viral NS3/4A protease has been well characterized, the mechanism for resistance of non-active site mutations is unclear. R155K and V36M often co-evolve and while R155K alters the electrostatic network at the binding site, V36M is more than 13 [A] away. In this study the mechanism by which V36M confers resistance, in the context of R155K, is elucidated with drug susceptibility assays, crystal structures, and molecular dynamics (MD) simulations for three protease inhibitors: telaprevir, boceprevir and danoprevir. The R155K and R155K/V36M crystal structures differ in the -2 helix and E2 strand near the active site, with alternative conformations at M36 and side chains of active site residues D168 and R123, revealing an allosteric coupling, which persists dynamically in MD simulations, between the distal mutation and the active site. This allosteric modulation validates the network hypothesis and elucidates how distal mutations confer resistance through propagation of conformational changes to the active site.

molecular biology↗