Biology subjects

Gomez-Dominguez, D.

Publications and source records attributed to Gomez-Dominguez, D..

8 recordsLinked to original sources

bioRxiv2026

Adenine base editing correction of LMNA c.745C>T (p.R249W) in congenital muscular dystrophy myoblasts improves cellular phenotype while revealing deleterious p.L248P bystander effects

BackgroundLMNA-related congenital muscular dystrophy (L-CMD) is a rare, life-threatening genetic disorder caused by point mutations in the LMNA gene, for which no effective treatment currently exists. It is characterized by early-onset muscle weakness, dropped-head syndrome, hypotonia, cardiac complications, and restrictive lung disease, frequently leading to premature death. The LMNA c.745C>T (p.R249W) mutation is the most prevalent amongst L-CMD patients. Given its monogenic nature, L-CMD represents a compelling candidate for gene therapy approaches. ResultsIn this study, we investigated the therapeutic potential of adenine base editing (ABE) to correct the pathogenic LMNA c.745C>T (p.R249W) mutation in human myoblasts. We evaluated multiple ABE variants and single-guide RNAs (sgRNAs), identifying optimal combinations that achieved efficient and specific correction of the mutant allele. However, we found that editing can also introduce an adjacent bystander mutation, c.743T>C (p.L248P). To determine the functional consequences of base editing, we established clonal cell lines reverted to wild type or harboring the p.L248P variant. Whereas wild-type edited cells showed a clear correction for all the studied parameters that were abnormal in R249W myoblasts, we found that L248P cells show nuclear abnormalities resembling those of R249W mutant cells, and their cellular function is partially compromised. These results demonstrate that ABE can effectively target the LMNA c.745C>T mutation but also reveal the significant impact of bystander edits on cellular physiology. ConclusionsOur findings provide proof-of-concept for the application of base editing as a therapeutic strategy for L-CMD, while underscoring the necessity of precise editing technologies to ensure both efficacy and safety in future clinical translation.

cell biology↗

bioRxiv2025

CRISPR/Cas9-mediated elimination of the LMNA c.745C>G pathogenic mutation enhances survival and cardiac function in LMNA-associated congenital muscular dystrophy

LMNA-associated congenital muscular dystrophy is a currently incurable rare genetic disorder characterized by early-onset muscle weakness, dilated cardiomyopathy and respiratory failure, resulting from mutations in the LMNA gene. In this study, we assessed the potential of a CRISPR-mediated strategy to eliminate the mutant allele Lmna c.745C>T, p.R249W using a mutation specific guide (sg745T). Results from R249W-mutation-carrying cellular models showed specific activity of the Cas9/sg745T complex towards the mutant allele. This property varied depending on the concentration of CRISPR components, with a loss of specificity observed with increased dosage. We tested this strategy in vivo using adeno-associated virus delivery in LmnaR249W mice. Despite being associated with a modest CRISPR activity, this therapeutic approach resulted in a 10% increase in the survival of R249W homozygous mice. Interestingly, a similar CRISPR activity improved the cardiac pathology developed by Lmna+/R249W animals, significantly extending their median survival. These results represent the first therapeutic validation of a CRISPR/Cas9-mediated gene editing strategy for the treatment of LMNA-associated congenital muscular dystrophy.

bioengineering↗

bioRxiv2024

CRISPR targeting of FOXL2 c.402C>G mutation reduces malignant phenotype in granulosa tumor cells and identifies anti-tumoral compounds

FOXL2 is a transcription factor essential for sex determination and ovary development and maintenance. Mutations in this gene are implicated in syndromes involving premature ovarian failure and granulosa cell tumors (GCTs). This rare cancer accounts for less than 5% of diagnosed ovarian cancers and is causally associated with the FOXL2 c.402C>G, p.C134W mutation in 97% of the adult cases (AGCTs). In this study, we employed CRISPR technology to specifically eliminate the FOXL2 c.402C>G mutation in granulosa tumor cells. Our results show that this Cas9-mediated strategy selectively targets the mutation without affecting the wild-type allele. Granulosa cells lacking the FOXL2 c.402C>G exhibit a reduced malignant phenotype, with significant changes in cell proliferation, invasion, and cell death. Furthermore, these modified cells are more susceptible to Dasatinib and Ketoconazole. Transcriptomic and proteomic analyses reveal that CRISPR-modified granulosa tumor cells shift their expression profiles towards a wild-type like phenotype. Additionally, this altered expression signature has led to the identification of new compounds with antiproliferative and pro-apoptotic effects on granulosa tumor cells. Our findings demonstrate the potential of CRISPR technology for the specific targeting and elimination of a mutation causing granulosa cell tumors, highlighting its therapeutic promise for treating this rare ovarian cancer. Simple SummaryAdult granulosa cell tumors (AGCTs), characterized by a specific point mutation (C134W) in the gene FOXL2, are less than 5% of all the diagnosed ovarian cancers. Surgery is the cornerstone treatment for AGCT even at relapse, with systemic therapy showing poor results. The aim of our study is to explore the potential therapeutic effect of the elimination of the C134W mutation. To achieve our goal, we have eliminated the mutant allele using CRISPR technology. Our results demonstrate that CRISPR-mediated elimination of FOXL2-C134W reduces the malignant phenotype of granulosa tumor cells, which change their transcriptional, proteomic, and cellular phenotype to a wild type like, granulosa type. Moreover, the induced changes allowed us to find new compounds with antitumoral activity. This work highlights the therapeutic potential of CRISPR mediated technology for the treatment of AGCT.

cancer biology↗

bioRxiv2021

Targeting the mitochondrial RNA methyltransferase TRMT61B reveals new therapeutic opportunities in aneuploid cancer cells

Chromosomal instability (CIN) is an important source of genetic and phenotypic variation that has been extensively reported as a critical cancer related property that improves tumor cell adaptation and survival. CIN and its immediate consequence, aneuploidy, provoke adverse effects on cellular homeostasis that need to be overcome by developing efficient anti-stress mechanisms. Perturbations in these safeguard responses might be detrimental for cancer cells and represent an important tumor specific Achilles heel since CIN and aneuploidy are very rare events in normal cells. On the other hand, epitranscriptomic marks catalyzed by different RNA modifying enzymes have been found to change under several stress insults. Although CIN and aneuploidy are important intracellular stressors, their biological connection with RNA modifications is pending to be determined. In an in silico search for new cancer biomarkers, we have identified TRMT61B, a mitochondrial RNA methyltransferase enzyme, to be associated with high levels of aneuploidy. In the present work, we study the connection of this molecule with cancer and aneuploidy. First, we show increased protein amounts of TRMT61B in tumor cell lines with imbalanced karyotype as well as in different tumor types compared to unaffected control tissues. In addition, we demonstrate that depletion of TRMT61B in melanoma cells reduces cell proliferation either by fostering apoptosis and inhibiting autophagy in high-aneuploid (ANEhigh) cells or by inducing senescence in the case of low-aneuploid (ANElow) cell lines. Further, TRMT61B elimination compromises mitochondrial function and reduces the expression of several mitochondrial encoded proteins that are part of the electron transport chain. Finally, transwell and xenograft experiments revealed a reduced invasive and tumorigenic capacity upon TRMT61B depletion that strengthen the therapeutic value of this aneuploidy-associated biomarker. These results, which connect tumorigenesis, aneuploidy and mitochondrial RNA methylation, bring to the cancer field a new putative strategy to specifically target high aneuploid tumors.

cancer biology↗

bioRxiv2021

Sublayer- and cell-type-specific neurodegenerative transcriptional trajectories in hippocampal sclerosis

Hippocampal sclerosis, the major neuropathological hallmark of temporal lobe epilepsy, is characterized by different patterns of neuronal loss. The mechanisms of cell-type specific vulnerability, their progression and histopathological classification remain controversial. Here using single-cell electrophysiology in vivo and immediate early gene expression, we reveal that superficial CA1 pyramidal neurons are overactive in epileptic rats and mice in vivo. Bulk tissue and single-nucleus expression profiling disclosed sublayer-specific transcriptomic signatures and robust microglial pro-inflammatory responses. Transcripts regulating neuronal processes such as voltage-channels, synaptic signalling and cell adhesion molecules were deregulated by epilepsy differently across sublayers, while neurodegenerative signatures primarily involved superficial cells. Pseudotime analysis of gene expression in single-nuclei and in situ validation revealed separated trajectories from health to epilepsy across cell types, and identified a subset of superficial cells undergoing a later stage in neurodegeneration. Our findings indicate sublayer- and cell type-specific changes associated with selective CA1 neuronal damage contributing to progression of hippocampal sclerosis.

neuroscience↗

bioRxiv2018

KCC2 regulates neuronal excitability and hippocampal activity via interaction with Task-3 channels

The K+/Cl- co-transporter KCC2 (SLC12A5) regulates neuronal transmembrane chloride gradients and thereby controls GABA signaling in the brain. KCC2 downregulation is observed in several neurological and psychiatric disorders including epilepsy, neuropathic pain and autism spectrum disorders. Paradoxical, excitatory GABA signaling is usually assumed to contribute to abnormal network activity underlying the pathology. We tested this hypothesis and explored the functional impact of chronic KCC2 downregulation in the rat dentate gyrus. Although the reversal potential of GABAA receptor currents was depolarized in KCC2 knockdown neurons, this shift was fully compensated by depolarization of their resting membrane potential. This effect was due to downregulation of Task-3 leak potassium channels that we show require KCC2 for membrane trafficking. Increased neuronal excitability upon KCC2 suppression altered dentate gyrus rhythmogenesis that could be normalized by chemogenetic hyperpolarization. Our data reveal KCC2 downregulation engages complex synaptic and cellular alterations beyond GABA signaling that concur to perturb network activity, thus offering novel targets for therapeutic intervention.

neuroscience↗

bioRxiv2018

Proximodistal organization of the CA2 hippocampal area

The proximodistal axis is considered a major organizational principle of the hippocampus. Interfacing between the hippocampus and other brain systems, the CA2 region apparently breaks this rule. Apart from its specific role in social memory, CA2 has been involved in temporal and contextual memory but mechanisms remain elusive. Here, we used intracellular and extracellular recordings followed by neurochemical identification of single-cells to evaluate CA2 and surrounding areas in the rat. We found marked proximodistal trends of synaptic activity, as well as in subthreshold membrane potentials and phase-locked firing coupled to theta and gamma oscillations. Opposite proximodistal correlations between membrane potential fluctuations and theta sinks and sources at different layers revealed influences from up to three different generators. CA2 memory engrams established after a social memory task reflected these trends. We suggest that the structure and function of CA2 is segregated along the proximodistal hippocampal axis.

neuroscience↗

bioRxiv2018

Dynamic embedding of salience coding in hippocampal spatial maps

Hippocampal CA1 neurons participate in dynamic ensemble codes for space and memory. Prominent features of the environment are represented by an increased density of place cells, but cellular principles governing the formation and plasticity of such disproportionate maps are unknown. We thus imaged experience-dependent long-term changes in spatial representations at the cellular level in the CA1 deep sublayer in mice learning to navigate in a virtual-reality environment. The maps were highly dynamic but gradually stabilized as over-representations for motivational (reward) and environmental (landmark) salience emerged in different time courses by selective consolidation of relevant spatial representations. Relocation of the reward extensively reorganized pre-formed maps by a mechanism involving rapid recruitment of cells from the previous location followed by their re-stabilization, indicating that a subset of neurons encode reward-related information. The distinct properties of these CA1 cells may provide a substrate by which salient experience forms lasting and adaptable memory traces.

neuroscience↗