Biology subjects

Thrasher, A. J.

Publications and source records attributed to Thrasher, A. J..

7 recordsLinked to original sources

bioRxiv2026

In-Depth Characterization of Stem Cell Potency and Genotoxicity for Clinical-Scale Ex Vivo CRISPR/Cas9 Gene Editing

Translating CRISPR/Cas9-based homology-directed repair (HDR) strategies into clinical application remains a major challenge due to limited standardization, concerns over safety, and efficacy issues. Here, we present a comprehensive and clinically compliant preclinical framework for the ex vivo correction of Wiskott-Aldrich Syndrome (WAS) using a CRISPR/Cas9-AAV6 platform targeting hematopoietic stem and progenitor cells (HSPCs). In this study, we established a clinical-compatible platform enabling large-scale manufacturing while preserving HSPC viability, stemness, and multilineage functionality. To overcome low HSPC long-term engraftment, we fine-tuned AAV dosing and transiently modulated p53 signalling, achieving significantly improved in vivo correction and repopulation. Importantly, we implemented a multi-tiered genotoxicity assessment strategy, integrating in silico, genome-wide, and orthogonal assays, revealing a largely favorable safety profile with minimal off-target risks and no signs of clonal dominance or transformation. Longitudinal in vivo safety monitoring revealed donor specific rare off-target events and structural variants. This highlights the crucial importance of patient monitoring after transplantation, further emphasized by the identification of a de novo chromosomal rearrangements that could be detected exclusively following cell engraftment in mice. This work offers a robust and adaptable roadmap for future HDR-based gene editing platforms, establishing critical benchmarks for efficacy, safety, and regulatory readiness in the development of advanced therapeutic medicinal products.

molecular biology↗

bioRxiv2023

CRISPR/Cas9-based disease modelling and functional correction of Interleukin 7 Receptor alpha Severe Combined Immunodeficiency in T-lymphocytes and hematopoietic stem cells

Interleukin 7 Receptor Severe Combined Immunodeficiency (IL7R-SCID) is a life-threatening disorder caused by homozygous mutations in the IL7RA gene. Defective IL7R expression in humans hampers T cell precursors proliferation and differentiation during lymphopoiesis resulting in absence of T cells in newborns, who succumb to severe infections and death early after birth. Previous attempts to tackle IL7R-SCID by viral gene therapy have shown that unregulated IL7R expression predisposes to leukaemia, suggesting the application of targeted gene editing to insert a correct copy of the IL7RA gene in its genomic locus and mediate its physiological expression as a more feasible therapeutic approach. To this aim, we have first developed a CRISPR/Cas9-based IL7R-SCID disease modelling system that recapitulates the disease phenotype in primary human T cells and hematopoietic stem and progenitor cells (HSPCs). Then, we have designed a knock-in strategy that targets IL7RA exon 1 and introduces via homology directed repair a corrective, promoterless IL7RA cDNA followed by a reporter cassette through AAV6 transduction. Targeted integration of the corrective cassette in primary T cells restored IL7R expression and rescued functional downstream IL7R signalling. When applied to HSPCs further induced to differentiate into T cells in an Artificial Thymic Organoid system, our gene editing strategy overcame the T cell developmental block observed in IL7R-SCID patients, while promoting full maturation of T cells with physiological and developmentally regulated IL7R expression. Finally, genotoxicity assessment of the CRISPR/Cas9 platform in HSPCs using biased and unbiased technologies confirmed the safety of the strategy, paving the way for a new, efficient, and safe therapeutic option for IL7R-SCID patients.

molecular biology↗

bioRxiv2022

Assessment of pre-clinical liver models based on their ability to predict the liver-tropism of AAV vectors

The liver is a prime target for in vivo gene therapies using recombinant adeno-associated viral vectors (rAAV). Multiple clinical trials have been undertaken for this target in the past 15 years, however we are still to see market approval of the first liver-targeted AAV-based gene therapy. Inefficient expression of the therapeutic transgene, vector-induced liver toxicity and capsid, and/or transgene-mediated immune responses reported at high vector doses are the main challenges to date. One of the contributing factors to the insufficient clinical outcomes, despite highly encouraging preclinical data, is the lack of robust, biologically- and clinically-predictive preclinical models. To this end, this study reports findings of a functional evaluation of six AAV vectors in twelve preclinical models of the human liver, with the aim to uncover which model is the most relevant for the selection of AAV capsid variant for safe and efficient transgene delivery to primary human hepatocytes. The results, generated by studies in models ranging from immortalized cells, iPSC-derived and primary hepatocytes, and primary human hepatic organoids to in vivo models, increased our understanding of the strengths and weaknesses of each system. This should allow the development of novel gene therapies targeting the human liver.

bioengineering↗

bioRxiv2022

Single Nucleus Sequencing of Human Colon Visceral Smooth Muscle Cells, PDGFRα Cells, and Interstitial Cells of Cajal

Background and AimsSmooth muscle cells (SMCs), Interstitial cells of Cajal (ICCs), and PDGFR+ cells (PCs) form a functional syncytium in the bowel known as the SIP syncytium. The SIP syncytium works in concert with the enteric nervous system (ENS) to coordinate bowel motility. However, our understanding of individual cell types that form this syncytium and how they interact with each other remains limited, with no prior single cell RNAseq analyses focused on human SIP syncytium cells. MethodsWe analyzed single-nucleus RNA sequencing data from 10,749 human colon SIP syncytium cells (5572 SMC, 372 ICC, and 4805 PC nuclei) derived from 15 individuals. ResultsConsistent with critical contractile and pacemaker functions and with known ENS interactions, SIP syncytium cell types express many ion channels including mechanosensitive channels in ICCs and PCs. PCs also prominently express ECM-associated genes and the inhibitory neurotransmitter receptor for vasoactive intestinal peptide (VIPR2), a novel finding. We identified two PC clusters that differ in expression of many ion channels and transcriptional regulators. Interestingly, SIP syncytium cells co-express 6 transcription factors (FOS, MEIS1, MEIS2, PBX1, SCMH1, and ZBTB16) that may be part of a combinatorial signature that specifies these cells. Bowel region-specific differences in SIP syncytium gene expression may correlate with regional differences in function, with right (ascending) colon SMCs and PCs expressing more transcriptional regulators and ion channels than SMCs and PCs in left (sigmoid) colon. ConclusionThese studies provide new insights into SIP syncytium biology that may be valuable for understanding bowel motility disorders and lead to future investigation of highlighted genes and pathways. SynopsisIn this first single nucleus RNASeq analysis of human SIP syncytium, we identify novel features of SIP syncytium cells, including two types of PDGFR+ cells, a SIP-specific combinatorial transcription factor signature, and colon region differences in gene expression.

cell biology↗

bioRxiv2022

Optimized cell culture conditions promote ex-vivo manipulation and expansion of primitive hematopoietic stem cells for therapeutic gene editing.

During the last few years, gene editing has emerged as a powerful tool for the therapeutic correction of monogenic diseases. CRISPR/Cas9 applied to hematopoietic stem and progenitor cells (HSPCs) has shown great promise in proof-of-principle preclinical studies to treat haematological disorders, and clinical trials using these tools are now underway. Nonetheless, there remain important challenges that need to be addressed, such as the efficiency of targeting primitive, long-term repopulating HSPCs and expand them in vitro for clinical purposes. Here we have tested the effect exerted by different culture media compositions on the ability of HSPCs to proliferate and undergo homology directed repair-mediated knock-in of a reporter gene, while preserving their stemness features during ex-vivo culture. We tested different combinations of compounds and demonstrated that by supplementing the culture media with inhibitors of histone deacetylases, and/or by fine-tuning its cytokine composition it is possible to achieve high levels of gene targeting in long-term repopulating HSPCs both in vitro and in vivo, with a beneficial balance between preservation of stemness and cell expansion, thus allowing to obtain a significant amount of edited, primitive HSPCs compared to established, state-of-the-art culture conditions. Overall, the implantation of this optimized ex vivo HSPC culture protocol will improve the efficacy, feasibility and applicability of gene editing and will likely provide one step further to unlock the full therapeutic potential of such powerful technology.

bioengineering↗

bioRxiv2020

Wiskott Aldrich syndrome protein regulates non-selective autophagy and mitochondrial homeostasis in human myeloid cells

The actin cytoskeletal regulator Wiskott Aldrich syndrome protein (WASp) has been implicated in maintenance of the autophagy-inflammasome axis in innate murine immune cells. Here, we show that WASp deficiency is associated with impaired rapamycin-induced autophagosome formation and trafficking to lysosomes in primary human monocyte-derived macrophages (MDMs). WASp reconstitution in vitro and in WAS patients following clinical gene therapy restores autophagic flux and is dependent on the actin-related protein complex ARP2/3. Induction of mitochondrial damage with CCCP, as a model of selective autophagy, also reveals a novel ARP2/3-dependent role for WASp in formation of sequestrating actin cages and maintenance of mitochondrial network integrity. Furthermore, mitochondrial respiration is suppressed in WAS patient MDMs and unable to achieve normal maximal activity when stressed, indicating profound intrinsic metabolic dysfunction. Taken together, we provide evidence of new and important roles of human WASp in autophagic processes and immunometabolic regulation, which may mechanistically contribute to the complex WAS immunophenotype.

physiology↗

bioRxiv2018

EROS is required for phagocyte NADPH oxidase function in humans and its deficiency causes Chronic Granulomatous Disease.

The phagocyte respiratory burst is mediated by the phagocyte NADPH oxidase, a multi-protein subunit complex that facilitates production of reactive oxygen species and which is essential for host defence. Monogenic deficiency of individual subunits leads to chronic granulomatous disease (CGD), which is characterized by an inability to make reactive oxygen species, leading to severe opportunistic infections and auto-inflammation. However, not all cases of CGD are due to mutations in previously identified subunits. We recently showed that Eros, a novel and highly conserved ER-resident transmembrane protein, is essential for the phagocyte respiratory burst in mice because it is required for expression of gp91phox-p22phox heterodimer, which are the membrane bound components of the phagocyte NADPH oxidase. We now show that the function of EROS is conserved in human cells and describe a case of CGD secondary to a homozygous EROS mutation that abolishes EROS protein expression. This work demonstrates the fundamental importance of EROS in human immunity and describes a novel cause of CGD.\n\nClinical ImplicationsChronic granulomatous disease is caused by an inability to make reactive oxygen species via the phagocyte NADPH oxidase. Mutations in C17ORF62/EROS, which controls gp91phox- p22phox abundance, are a novel cause of chronic granulomatous disease.\n\nKey MessagesO_LIThe murine gene, Eros, is known to regulate abundance of gp91phox-p22phox heterodimer and Eros deficient mice are susceptible to infection\nC_LIO_LIWe show that the function of EROS is conserved in human cells and that a homozygous mutation in EROS causes chronic granulomatous disease\nC_LI

immunology↗