Biology subjects

Hautala, T.

Publications and source records attributed to Hautala, T..

2 recordsLinked to original sources

bioRxiv2024

T cell correction pipeline for Inborn Errors of Immunity

CRISPR/Cas9 gene editing technology is a promising tool for correcting pathogenic variants for autologous cell therapies for Inborn Errors of Immunity (IEI). The present IEI correction strategies mainly focus on the knock-in of therapeutic cDNAs, or knockout of the disease-causing gene when feasible. These strategies address many single-gene defects but may disrupt gene expression and require significant optimization for each newly discovered IEI-causing gene, highlighting the need for complementary platforms that can precisely correct diverse pathogenic variants. Here, we present a safe and efficient T cell single nucleotide variant (SNV) correction pipeline based on homology-directed repair (HDR), suitable for diverse monogenic mutations. By using founder mutations of Deficiency of ADA2 (DADA2), Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) and Cartilage Hair Hypoplasia (CHH) as IEI models, we show that our pipeline can achieve up to 80% bi-allelic editing, with resultant functional correction of the disease phenotype in patient T cells. We do not find detectable pre-malignant off-target effects or karyotypic, transcriptomic or proteomic aberrations upon profiling patient T cells with GUIDE-seq, single cell RNA sequencing, PacBio based long-read whole genome sequencing, and high-throughput proteomics. This study demonstrates that HDR-based SNV editing is a safe and effective option for IEI T cell correction and that it could be developed to an autologous T cell therapy, as the presented protocol is scalable for a GMP-compatible workflow. This study is a step towards the development of gene correction platform that targets a broad number of monogenic mutations. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/610811v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1f1eb47org.highwire.dtl.DTLVardef@18dbcc2org.highwire.dtl.DTLVardef@63862dorg.highwire.dtl.DTLVardef@1fe1561_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT C_FIG

bioengineering↗

bioRxiv2018

Haploinsufficiency of A20 impairs protein-protein interactome and leads into caspase-8-dependent enhancement of NLRP3 inflammasome activation

ObjectivesTNFAIP3 encodes A20 that negatively regulates nuclear factor kappa light chain enhancer of activated B cells (NF-{kappa}B), the major transcription factor coordinating inflammatory gene expression. TNFAIP3 polymorphisms have been linked with a spectrum of inflammatory and autoimmune diseases and recently, loss-of-function mutations in A20 were found to cause a novel inflammatory disease haploinsufficiency of A20 (HA20). Here we describe a family with HA20 caused by a novel TNFAIP3 loss-of-function mutation and elucidate the upstream molecular mechanisms linking HA20 to dysregulation of NF-{kappa}B and the related inflammasome pathway.\n\nMethodsNF-{kappa}B activation was studied in a mutation-expressing cell line using luciferase reporter assay. Physical and close-proximity protein-protein interactions of wild-type and TNFAIP3 p.(Lys91*) mutant A20 were analyzed using mass spectrometry. NF-{kappa}B -dependent transcription, cytokine secretion, and inflammasome activation were compared in immune cells of the HA20 patients and control subjects.\n\nResultsThe protein-protein interactome of p.(Lys91*) mutant A20 was severely impaired, including inter-actions with proteins regulating NF-{kappa}B activation, DNA repair responses, and the NLR family pyrin domain containing 3 (NLRP3) inflammasome. The p.(Lys91*) mutant A20 failed to suppress NF-{kappa}B signaling, which led to increased NF-{kappa}B -dependent proinflammatory cytokine transcription. Functional experiments in the HA20 patients immune cells uncovered a novel caspase-8-dependent mechanism of NLRP3 inflammasome hyperresponsiveness that mediated the excessive secretion of interleukin-1{beta} and -18.\n\nConclusionsThe current findings significantly deepen our understanding of the molecular mechanisms underlying HA20 and other diseases associated with reduced A20 expression or function, paving the way for future therapeutic targeting of the pathway.

molecular biology↗