Biology subjects

Halloy, F.

Publications and source records attributed to Halloy, F..

3 recordsLinked to original sources

bioRxiv2024

miRNA-29-CLIP uncovers new targets and functions to improve skin repair

MicroRNAs (miRNAs) control organogenesis in mammals but their role in specific cell types is not fully explored. miRNAs exert their function by binding mRNAs and inhibiting translation. Skin is an excellent model to study the role of miRNAs in primary cells of epidermal (keratinocytes) and mesodermal (fibroblasts) origin, because the growth of these cells is tightly controlled at translation. Previous research demonstrated that miRNA-29 family functions during skin repair, however, the exact mRNA targets and the downstream mechanisms of miRNA-29-mediated regulation of cell growth is missing. Here, we use miRNA crosslinking and immunoprecipitation (miRNA-CLIP) method to find the direct targets of miRNA-29 in keratinocytes and fibroblasts from human skin. We uncover previously unrecognized roles of miRNA-29 in protein folding and RNA processing, common to all cell types tested, and determine the cell-specific role of miRNA-29. Using modified anti-sense oligonucleotides (ASO) in 2D and 3D cultures of keratinocytes and fibroblasts, we enhanced cell-to-matrix adhesion and found an autocrine and paracrine mechanism of miRNA-29-dependent cell growth. Our results include a comprehensive list and functional analyses of mRNAs directly bound by miRNA-29 keratinocytes and fibroblasts, determined by miRNA-CLIP and ASO-mediated inhibition of miRNA-29 followed by RNA-seq. We reveal a full transcriptome of human keratinocytes with enhanced adhesion to the wound matrix, which supports regeneration of the epidermis and is regulated by miRNA-29. The functions of miRNA-29 identified in this study can provide a new approach to improve cutaneous repair by restoring and enhancing the endogenous mechanisms through the stage-specific delivery of miRNA-29 ASO.

cell biology↗

bioRxiv2024

Systemic delivery of a splice-switching oligonucleotide heteroduplex corrects splicing in central nervous system and muscle in spinal muscular atrophy mice

Oligonucleotide therapeutics are an established class of drugs for the treatment of genetic disorders. Their clinical development is challenging, however, as they typically distribute poorly to extra-hepatic tissues after systemic injection. Here we tested the heteroduplex oligonucleotide (HDO) platform for systemic delivery of SMN2 splice-switching oligonucleotides of 2-O-methoxyethyl phosphorothioate or phosphorodiamidate morpholino oligomer chemistries. We first showed that splice-switching HDO cargoes correct SMN2 splicing in cells derived from spinal muscular atrophy (SMA) patients, and validated extra-hepatic activity in spinal cord and muscle in a mouse model of SMA following systemic delivery. Our study raises prospects for delivery of nusinersen, the 2-O-methoxylethyl phosphorothioate oligonucleotide therapy approved for SMA and currently delivered by intrathecal injection, by systemic injection exploiting the HDO chemistry platform. Our findings also suggest that oligonucleotide drugs lacking convincing in vivo efficacy in muscle tissue could be delivered effectively by the HDO technology.

molecular biology↗

bioRxiv2020

Delivery of oligonucleotides to bone marrow to modulate ferrochelatase splicing in a mouse model of Erythropoietic Protoporphyria

Erythropoietic protoporphyria (EPP) is a rare genetic disease in which patients experience acute phototoxic reactions after sunlight exposure. It is caused by a deficiency in ferrochelatase (FECH) in the heme biosynthesis pathway. Most patients exhibit a loss-of-function mutation in trans to an allele bearing a SNP that favours aberrant splicing of transcripts. One viable strategy for EPP is to deploy splice-switching oligonucleotides (SSOs) to increase FECH synthesis, whereby an increase of a few percent would provide therapeutic benefit. However, successful application of SSOs in bone marrow cells is not described. Here, we show that SSOs comprising methoxyethyl-chemistry increase FECH levels in cells. We conjugated one SSO to three prototypical targeting groups and administered them to a mouse model of EPP in order to study their biodistribution, their metabolic stability and their FECH splice-switching ability. The SSOs exhibited distinct distribution profiles, with increased accumulation in liver, kidney, bone marrow and lung. However, they also underwent substantial metabolism, mainly at their linker groups. An SSO bearing a cholesteryl group increased levels of correctly spliced FECH transcript by 80% in the bone marrow. The results provide a promising approach to treat EPP and other disorders originating from splicing dysregulation in the bone marrow.

molecular biology↗