Biology subjects

Lopez-Martinez, A.

Publications and source records attributed to Lopez-Martinez, A..

4 recordsLinked to original sources

bioRxiv2025

Characterisation of DMPK and MBNL1 expression in cell models of Myotonic Dystrophy: A platform for drug screening

Myotonic dystrophy type I (DM1) is caused by CTG repeat expansions in the DMPK gene leading to mRNA toxicity and sequestration of the splicing regulator MBNL1, affecting many tissues. We have developed an in vitro screening platform based on ddPCR and in-cell western to quantify these mRNAs and proteins and characterised more than 20 cell models to define DM1 biomarkers that could be useful for drug screening. DMPK protein levels were reduced in DM1-immortalised myoblasts and myotubes, but not in fibroblasts, while MBNL1 protein was consistently lower in all DM1 myogenic cultures, whether primary or immortalised. Myogenic differentiation of cultures led to an increase in DMPK mRNA expression, which was translated into increased MBNL1 sequestration in foci. We further corroborated the platforms ability to assess therapeutic outcomes, evaluating the effect of a DMPK gapmer ASO and one siRNA: while the gapmer increased MBNL1 protein levels, the siRNA had no significant effect on MBNL1 release. Our platform and the in-depth characterisation of some of the most used models would be of use to the DM1 research community. Significance statementMyotonic dystrophy type I (DM1) is a multisystemic disease with a complex pathogenesis and multiple outcome measures for drug assessment in vitro. In the last years, the increasing number of new potential therapies targeting DM1 in clinical trials has increased the need for robust and rapid evaluation of preclinical candidates, as well as in-depth knowledge of the cell models used. Here, we present a new cell-based platform that enables robust quantification of DMPK and MBNL1 in cell culture for cell model characterisation and drug screening. Indeed, we highlight the differences observed in DMPK and MBNL1 protein quantification in primary fibroblasts and myotubes, immortalised fibroblasts, myoblasts and myotubes. We then, we performed a proof-of-concept drug evaluation of potential therapeutic strategies targeting DMPK, showing the most suitable for targeting the DMPK expanded transcript.

cell biology↗

bioRxiv2024

Collablots: Quantification of collagen VI levels and its structural disorganisation in cell cultures from patients with collagen VI-related dystrophies

AimsThis study aims to develop a quantitative method for assessing collagen VI expression in cell cultures, which is crucial for the diagnosis and treatment of collagen VI-related dystrophies. MethodsWe developed a combined in-cell western (ICW) and on-cell western (OCW) assay, that we have called collablot to quantify collagen VI and its organisation in the extracellular matrix of cell cultures from patients and healthy controls. To optimise it, we optimised cell density and the protocols to induce collagen expression in cultures, as well as the cell fixation and permeabilisation methods. This was completed with a thorough selection of collagen antibodies and a collagen hybridising peptide (CHP). We then used collablots to compare cultures from patients and controls and evaluate therapeutic interventions in the cultures. ResultsCollablots enabled the quantification of collagen VI expression in both control and patient cells, aligning with immunocytochemistry findings and detecting variations in collagen VI expression following treatment of the cultures. Additionally, CHP analysis revealed a marked increase in collagen network disruption in patients compared to the controls. ConclusionsThe collablot assay represents an optimal method for quantifying collagen VI expression and its organisation in culture and assessing the effect of therapies. Key Points- Evaluating therapies for collagen VI-related dystrophies (COL6-RD) requires the quantification of collagen VI levels. - Collablot assays are a novel method for quantifying collagen VI expression and its structural organisation in cell culture. - Due to the significant role of phenotype heterogeneity in this complex disease, quantifying collagen alone might not be adequate for diagnosing COL6-RD, but the addition of a peptide to quantify collagen disorganisation could help in the characterisation of patient cultures.

neuroscience↗

bioRxiv2023

Deletion of exons 45 to 55 in the DMD gene: from the therapeutic perspective to the in vitro model

Gene editing therapies in development for correcting out-of-frame DMD mutations in Duchenne muscular dystrophy aim to replicate benign spontaneous deletions. Deletion of 45-55 DMD exons (del45-55) was described in asymptomatic subjects, but recently serious skeletal and cardiac complications have been reported. Uncovering why a single mutation like del45-55 is able to induce diverse phenotypes and grades of severity may impact the strategies of emerging therapies. Cellular models are essential for this purpose, but their availability is compromised by scarce muscle biopsies. Here, we have introduced through CRISPR-Cas9 edition, a del45-55 mimicking the intronic breakpoints harboured by a subset of patients of this form of dystrophinopathy, into a Duchenne patients cell line. Dystrophin expression was restored in edited myoblasts and the myogenic defects were ameliorated. Besides confirming the potential of CRISPR-Cas9 to create tailored mutations as a useful approach to generate in vitro models, we also generated an immortalized myoblast line derived from a patient with a specific del45-55. Overall, we provide helpful resources to deepen into unknown factors responsible for DMD-pathophysiology. SUMMARY STATEMENTWe restored dystrophin expression in a DMD culture by replicating the exact deletion in exons 45-55 harboured by mild patients, testing this therapeutic approach, and creating a new cell model.

cell biology↗

bioRxiv2020

A CRISPR/Cas9-Based Approach For Editing Immortalised Human Myoblasts To Model Duchenne Muscular Dystrophy In Vitro

Gene edition methods are an attractive putative therapeutic option for Duchenne muscular dystrophy and they have an immediate application in the generation of research models. To generate two new edited myoblast cultures that could be useful in vitro drug screening, we have optimised a CRISPR/Cas9 gene edition protocol. We have successfully used it in wild type immortalised myoblasts to delete exon 52 of the dystrophin gene: DMD{Delta}52-Model, modelling a common Duchenne muscular dystrophy mutation; and in patients immortalised cultures we have deleted an inhibitory microRNA target region of the utrophin UTR, leading to utrophin upregulation. We have characterised these cultures and, to show their use in the assessment of DMD treatments, we have performed exon skipping in the DMD{Delta}52-Model and have used the unedited cultures/ DMD-UTRN-Model combo to assess utrophin overexpression after drug treatment. While the practical use of DMD{Delta}52-Model is limited to the validation to our gene edition protocol, DMD-UTRN-Model offers a possible therapeutic gene edition target as well as a useful positive control in the screening of utrophin overexpression drugs.

neuroscience↗