Biology subjects

Charrier, A.

Publications and source records attributed to Charrier, A..

3 recordsLinked to original sources

bioRxiv2023

Prevalence study of cellular capsid-specific immune responses to AAV1, 2, 4, 5, 8, 9 and rh10 reveals particular features for AAV9

Recombinant adeno-associated virus (rAAV) vectors appear, more than ever, to be efficient viral vectors for in vivo gene transfer as illustrated by the approvals of 7 drugs across Europe and the USA. Nevertheless, pre-existing immunity to AAV capsid in humans remains one of the major limits for a successful clinical translation. Whereas pre-existing humoral response to AAV capsid is well documented, the prevalence of pre-existing capsid-specific T cell responses still needs to be studied and characterized. Here, we investigated the prevalence of AAV-specific circulating T cells towards AAV2, 4, 5, 8, 9 and rh10 in a large cohort of healthy donors using the standard IFN{gamma} ELISpot assay. We observed the highest prevalence of pre-existing cellular immunity to AAV9 serotype followed by AAV8, AAV4, AAV2, AAVrh10 and AAV5 independently of the donors serological status. An in-depth analysis of T cell responses towards the 2 most prevalent serotypes 8 and 9 shows that IFN{gamma} secretion is mainly mediated by CD8 T cells for both serotypes. A polyfunctional analysis reveals different cytokine profiles between AAV8 and AAV9. Surprisingly, no IL-2 secretion was mediated by anti-AAV9 immune cells suggesting that these cells may rather be exhausted or terminally differentiated than cytotoxic T cells. Altogether, these results suggest that pre-existing immunity to AAV may vary depending on the serotype and support the necessity of using multiparametric monitoring methods to better characterize anti-capsid cellular immunity and foresee its impact in rAAV-mediated clinical trials.

immunology↗

bioRxiv2023

Molecular regulation of PPARγ/RXRα signaling by the novel cofactor ZFP407

Cofactors interacting with PPAR{gamma} can regulate adipogenesis and adipocyte metabolism by modulating the transcriptional activity and selectivity of PPAR{gamma} signaling. ZFP407 was previously demonstrated to regulate PPAR{gamma} target genes such as GLUT4, and its overexpression improved glucose homeostasis in mice. Here, using a series of molecular assays, including protein-interaction studies, mutagenesis, and ChIP-seq, ZFP407 was found to interact with the PPAR{gamma}/RXR protein complex in the nucleus of adipocytes. Consistent with this observation, ZFP407 DNA binding sites significantly overlapped with PPAR{gamma} sites, with more than half of ZFP407 binding sites overlapping with PPAR{gamma} DNA binding sites. Transcription factor binding motifs enriched in these overlapping sites included GFY-Staf, ELF1, ETS, ELK1, and ELK4, which regulate key functions within adipocytes. Site-directed mutagenesis of frequent PPAR{gamma} phosphorylation or SUMOylation sites did not prevent its regulation by ZFP407, while mutagenesis of ZFP407 regions necessary for RXR and PPAR{gamma} binding abrogated any impact of ZFP407 on PPAR{gamma} activity. These data suggest that ZFP407 controls the activity of PPAR{gamma}, but does so independently of post-translational modifications, likely by direct binding, establishing ZFP407 as a newly identified PPAR{gamma} cofactor. In addition, ZFP407 was also found to bind to DNA in regions that did not overlap with PPAR{gamma}. These DNA binding sites were more significantly enriched for the transcription factor binding motifs of GFY and ZNF143, which may contribute to the non-PPAR{gamma} dependent functions of ZFP407 in adipocytes and other cell types.

molecular biology↗

bioRxiv2023

Physical mechanisms of red blood cell splenic filtration

The splenic interendothelial slits fulfill the essential function of continuously filtering red blood cells (RBCs) from the bloodstream to eliminate abnormal and aged cells. To date, the process by which 8 {micro}m RBCs pass through 0.3 {micro}m-wide slits remains enigmatic. Does the slit caliber increase during RBC passage as sometimes suggested? Here, we elucidated the mechanisms that govern the RBC retention or passage dynamics in slits by combining multiscale modeling, live imaging, and microfluidic experiments on an original device with sub-micron wide physiologically calibrated slits. We observed that healthy RBCs pass through 0.28 {micro}m-wide rigid slits at 37{degrees}C. To achieve this feat, they must meet two requirements. Geometrically, their surface area-to-volume ratio must be compatible with a shape in two tether-connected equal spheres. Mechanically, the cells with a low surface area-to-volume ratio (28 % of RBCs in a 0.4 {micro}m-wide slit) must locally unfold their spectrin cytoskeleton inside the slit. In contrast, activation of the mechanosensitive PIEZO1 channel is not required. The RBC transit time through the slits follows a -1 and -3 power law with in-slit pressure drop and slip width, respectively. This law is similar to that of a Newtonian fluid in a 2D Poiseuille flow, showing that the dynamics of RBCs is controlled by their cytoplasmic viscosity. Altogether, our results show that filtration through submicron-wide slits is possible without further slit opening. Furthermore, our approach addresses the critical need for in-vitro evaluation of splenic clearance of diseased or engineered RBCs for transfusion and drug delivery. Significance StatementSplenic filtration of red blood cells through narrow interendothelial slits remains poorly understood despite its physiological significance as experiments and imaging of red cells passing through the slits are lacking. Here, we coupled live imaging, biomimetic submicron-fluidics, and multiscale modeling to quantify passage conditions. Remarkably, healthy 8-{micro}m cells can pass through 0.28-{micro}m slits at body temperature. This event is conditioned to cells being able to deform into two tether-connected equal spheres and, in limiting cases, to unfold their spectrin cytoskeleton. We showed that cells behave like a Newtonian fluid and that their dynamics is controlled by the inner fluid viscosity. We thus propose an in-vitro and in-silico approach to quantify splenic clearance of diseased cells and cells engineered for transfusion and drug delivery.

biophysics↗