Biology subjects

Vazquez-Manrique, R. P.

Publications and source records attributed to Vazquez-Manrique, R. P..

3 recordsLinked to original sources

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↗

bioRxiv2021

Alternative strategy to induce CRISPR-mediated genetic changes in hematopoietic cells

Acute Myeloid Leukaemia is a complex heterogenous disease caused by clonal expansion of undifferentiated myeloid precursors. Recently, several haematological models have been developed with CRISPR/Cas9, using viral vectors, because blood cells are hard to transfect. To avoid virus disadvantages, we have developed a strategy to generate CRISPR constructs, by means of PCR, which any lab equipped with basic technology can implement. These PCR-generated constructs enter easily into hard-to-transfect cells. After testing its functionality by editing MYBL2 gene in HEK293 cells, we successfully introduced the R172 mutation in IDH2 gene in NB4 cells that expresses constitutively the Cas9 nuclease. Comparing our methodology with ribonucleoprotein strategies, we found that mutation introduction efficiency was similar between both methodologies, and no off-target events were detected. Our strategy represents a valid alternative to introduce desired mutations in hard to transfect leukemic cells, avoiding using huge vectors or viral transduction.

molecular biology↗

bioRxiv2018

Loss of glutathione redox homeostasis impairs proteostasis by inhibiting autophagy-dependent protein degradation

In the presence of aggregation-prone proteins, the cytosol and endoplasmic reticulum (ER) undergo a dramatic shift in their respective redox status, with the cytosol becoming more oxidized and the ER more reducing. However, whether and how changes in the cellular redox status may affect protein aggregation is unknown. Here, we show that C. elegans mutants lacking glutathione reductase gsr-1 gene enhance the deleterious phenotypes of heterologous human as well as endogenous worm aggregation-prone proteins. These effects are phenocopied by the GSH depleting agent diethyl maleate. Additionally, gsr-1 mutants abolish the nuclear translocation of HLH-30/TFEB transcription factor, a key inducer of autophagy, and strongly impair the degradation of the autophagy substrate p62/SQST-1::GFP, revealing glutathione reductase may have a role in the clearance of protein aggregates by autophagy. Blocking autophagy in gsr-1 worms expressing aggregation-prone proteins results in strong synthetic developmental phenotypes and lethality, supporting the physiological importance of glutathione reductase in the regulation of misfolded protein clearance. Furthermore, impairing redox homeostasis in both yeast and mammalian cells induces toxicity phenotypes associated with protein aggregation. Together, our data reveal that glutathione redox homeostasis may be central to proteostasis maintenance through autophagy regulation.

genetics↗