Biology subjects

El Khoury, R.

Publications and source records attributed to El Khoury, R..

3 recordsLinked to original sources

bioRxiv2025

Fully Human Stem Cell-Derived BBB Model for Clinical Disease Modeling and Neurotropic AAV selection

The blood-brain barrier (BBB) is a highly functionalized vascular interface which regulates the exchange of substances between the neural parenchyma and its periphery. BBB leakage, leading to its uncontrolled permeability, is increasingly recognized to facilitate the onset of neuropathologies and aggravate their clinical progression. In vitro models of the BBB have rapidly evolved into elaborated structures that mimic its spatial architecture and multicellular nature. However, their cellular components are currently highly heterogeneous in origin and maturation state. Here, we have developed novel procedures to establish reproducible and scalable sources of endothelial, mural and astroglial cells generating a fully human pluripotent stem cell (hPSC)-derived BBB model, termed thBBBA. hPSC-derived BBB cell types are readily assembled into thBBBAs that develop mature functional properties with high barrier impermeability. Mature thBBBAs can also be generated by frozen hPSC-derived cell samples, providing a simple and scalable off-the-shelf system for general use. thBBBAs were instrumental in identifying the critical pathological role of an IL-6 autocrine source in disrupting thBBBA integrity, increasing its permeability to NMDAR antibodies from autoimmune encephalitis patients, and revealing the therapeutic effects of tocilizumab in this setting. Additionally, we have shown that thBBBAs are an invaluable system for ranking the clinical readiness of novel engineered AAV neurotropic capsids, previously selected in animal models or in vitro systems.

neuroscience↗

bioRxiv2023

DNA i-motif levels are overwhelmingly depleted in living human cells: insights from in-cell NMR.

I-Motifs (iM) are non-canonical DNA structures potentially forming in accessible, single-stranded, cytosine-rich genomic regions, with regulatory roles. Chromatin, protein interactions, and intracellular properties seem to govern iM formation at sites with i-motif formation propensity (iMFPS) in human cells, yet their specific contributions remain unclear. Using in-cell NMR with oligonucleotide iMFPS models, we monitored iM-associated structural equilibria in asynchronous and cell cycle-synchronized HeLa cells at 37{degrees}C. Our findings show that iMFPS displaying pHT<7 under reference in vitro conditions occur predominantly in unfolded states in cells, while those with pHT >7 occur as a mix of folded and unfolded states depending on the cell cycle phase. Comparing these results with previous data obtained using an iM-specific antibody (iMab) revealed that cell cycle-dependent iM formation has a dual origin and iM formation concerns only a small fraction (possibly 1%) of genomic sites with iM formation propensity. We propose a comprehensive model aligning observations from iMab and in-cell NMR and enabling the identification of iMFPS capable of adopting iM structures under physiological conditions in living human cells. Our results suggest that many iMFPS may have biological roles linked to their unfolded states.

biophysics↗

bioRxiv2023

Fibroblasts-dependent maturation and phenotype exacerbation of dystrophic hiPSC-derived MYOtissues enables muscle strength evaluation for gene therapy screening

Current gene therapy approaches for Duchenne muscular dystrophy (DMD) using AAV-mediated delivery of microdystrophin ({micro}Dys) have shown limited efficacy in patients, contrasting with the favorable outcomes observed in animal models. This discrepancy is partly due to the lack of models that replicate key pathogenic features associated with the severity of the human disease, such as fibrosis and muscle dysfunction. To tackle the translational gap, we develop a human disease model that recapitulates these critical hallmarks of DMD for a more predictive therapeutic investigation. Using a muscle engineering approach, we generate MYOrganoids from iPSC-derived muscle cells co-cultured with fibroblasts that enable functional maturation for muscle force analysis upon contractions. Incorporation of DMD fibroblasts within DMD iPSC-derived muscle cells allows phenotypic exacerbation by unraveling of fibrotic signature and fatiguability through cell-contact-dependent communication. Although {micro}Dys gene transfer partially restores muscle resistance, it fails to fully restore membrane stability and reduce profibrotic signaling. These findings highlight the persistence of fibrotic activity post-gene therapy in our human DMD system, an unparalleled aspect in existing DMD models, and provide the opportunity to explore the underlying mechanisms of dysregulated cellular communication to identify anti-fibrotic strategies empowering gene therapy efficacy.

bioengineering↗