Biology subjects

Marchalot, A.

Publications and source records attributed to Marchalot, A..

2 recordsLinked to original sources

bioRxiv2020

Splice switching oligonucleotide mediated gene knockdown in B cells and plasma cells

The need to identify new therapeutic approaches to the treatment of cancers of the B lymphoid lineage is crucial. Unlike CRISPR/Cas technology, antisense strategies result in transient modifications of gene expression and lack mutagenic effects at the DNA level. Here, we provide evidence for efficient knockdown of c-REL and RELA expression after treatment with splice switching antisense oligonucleotides (SSO) inducing exon skipping and reading frameshifts. We also developed a tool to facilitate the choice of exons for on purpose inhibition of mouse and human gene expression. Interestingly, treatments with morpholino SSO targeting the c-REL exon 2 donor splice site or RELA exon 5 acceptor splice site elicited very efficient knockdown in diffuse large B cell lymphoma (DLBCL) cell lines and antibody-secreting cells derived from primary human B cells. Consistent with the clinical relevance of c-REL activation in DLBCLs, treatment with c-REL SSO induced major alterations in NF-{kappa}B and TNF signalling pathways and strongly decreased cell viability. Altogether, SSO-mediated knockdown is a powerful approach to transiently inhibit the expression of given genes in B-lineage cells that should pave the way for cancer treatments, provided optimized ligand-conjugations for in vivo delivery. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/302984v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@170de4forg.highwire.dtl.DTLVardef@113229corg.highwire.dtl.DTLVardef@145377forg.highwire.dtl.DTLVardef@13e8c1e_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2019

Uncoupling splicing from transcription using antisense oligonucleotides reveals a dual role for I exon donor splice sites in antibody class switching

Class switch recombination (CSR) changes antibody isotype by replacing C constant exons with different constant exons located downstream on the immunoglobulin heavy (IgH) locus. During CSR, transcription through specific switch (S) regions and processing of noncoding germline transcripts (GLTs) are essential for the targeting of Activation-Induced cytidine Deaminase (AID). While CSR to IgG1 is abolished in mice lacking I{gamma}1 exon donor splice site (dss), many questions remain regarding the importance of I exon dss recognition in CSR. To further clarify the role of I exon dss in CSR, we first evaluated RNA polymerase II (RNA pol II) loading and chromatin accessibility in S regions after activation of mouse B cells lacking I{gamma}1 dss. We found that deletion of I{gamma}1 dss markedly reduced RNA pol II pausing and active chromatin marks in the S{gamma}1 region. We then challenged the post-transcriptional function of I exon dss in CSR by using antisense oligonucleotides (ASO) masking I exon dss on GLTs. Treatment of stimulated B cells with an ASO targeting I{gamma}1 dss, in the acceptor S{gamma}1 region, or I dss, in the donor S region, did not decrease germline transcription but strongly inhibited constitutive splicing and CSR to IgG1. Altogether, this study reveals that the recognition of I exon dss first supports RNA pol II pausing and the opening of chromatin in targeted S regions and that GLTs splicing events using constitutive I exon dss appear mandatory for the later steps of CSR, most likely by guiding AID to S regions.

immunology↗