Biology subjects

Wi, D.

Publications and source records attributed to Wi, D..

5 recordsLinked to original sources

bioRxiv2026

Versatile electroporation protocols enable reproducible CRISPR-RNP delivery across multiple primary mouse cells of the hematopoietic lineage

Genetic engineering of primary hematopoietic cells is essential for mechanistic immunology studies, however the development of cell-based therapies yet remains constrained by two major factors: the fragility of many primary lineages and challenges of viral delivery platforms that are costly, time-intensive, and biologically confounding. Here, we optimized scalable, non-viral CRISPR-Cas9 electroporation workflows using the ExPERT platform across three primary mouse hematopoietic cell types: OT-I CD8 T cells, bone marrow-derived macrophages (BMDMs), and hematopoietic stem cells (HSCs). In activated OT-I CD8 T cells, two electroporation programs supported high-efficiency mRNA and RNP delivery with minimal impact on cell viability or proliferative capacity, with subtle activation-state-dependent sensitivity at higher energy settings. Extending optimization to myeloid and stem cell lineages, we found that BMDMs maintained high viability following electroporation, and a high-performing electroporation program supported robust RNP delivery and efficient target gene knockout while preserving macrophage differentiation. In HSCs, the same program enabled consistent RNP delivery, sustained viability, and reproducible gene knockout. Together, these findings establish ExPERT electroporation as a robust, reproducible, and modular platform for non-viral genome editing across primary mouse hematopoietic lineages, lowering barriers to rapid genetic perturbation for both discovery and translational applications.

cell biology↗

bioRxiv2025

Immunometabolic determinants of long-term response in leukemia patients receiving CD19 CAR T cell therapy

Although most patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) receiving CD19-targeted chimeric antigen receptor (CAR) T cell therapy achieve remission, loss of CAR T cell functionality and subsequent relapse remains an unmet therapeutic need. We applied an integrative approach to study the immunometabolism of pre- and post-infusion CD19-CAR T cells of patients with relapsed/refractory B-ALL. Pre-infusion CAR T cells of long-term responders (LTR) had increased oxidative phosphorylation, fatty acid oxidation, and pentose phosphate pathway activities, higher mitochondrial mass, tighter cristae, and lower mTOR expression compared to products of short-term responders. Post-infusion CAR T cells in bone marrow (BM) of LTR had high immunometabolic plasticity and mTOR-pS6 expression supported by the BM microenvironment. Transient inhibition of mTOR during manufacture induced metabolic reprogramming and enhanced anti-tumor activity of CAR T cells. Our findings provide insight into immunometabolic determinants of long-term response and suggest a therapeutic strategy to improve long-term remission.

immunology↗

bioRxiv2024

ABA-regulated JAZ1 Proteins Bind NAC42 Transcription Factors to Suppress the Activation of Phytoalexin Biosynthesis in Plants

Phytoalexins are plant defense metabolites whose biosynthesis remains suppressed until elicited by a pathogen or stress, yet the mechanism of their suppression has remained elusive. The transcription factor GmNAC42-1 is an important and direct activator of the biosynthesis of glyceollin phytoalexins in soybean. Yet, without elicitation, overexpressing GmNAC42-1 is insufficient to activate the expression of glyceollin biosynthetic genes, suggesting that the activity of GmNAC42-1 may be suppressed by a negative regulator. JAZ1 proteins are negative regulators of the canonical jasmonic acid (JA) signaling pathway. JAZ protein degradation and JAZ gene transcription comprise antagonistic mechanisms that activate and suppress JA signaling, respectively. In search for negative regulators of glyceollin biosynthesis, we identified by RNA-seq analysis abscisic acid (ABA) signaling and GmJAZ1 genes that are oppositely regulated compared to glyceollin biosynthesis. Long-term ABA treatment upregulated GmJAZ1 transcripts, whereas its biosynthesis inhibitor fully suppressed their upregulation by dehydration stress. Opposite patterns were observed for glyceollin biosynthesis. RNAi silencing of GmJAZ1s prevented the suppression of glyceollin biosynthesis by dehydration and derepressed glyceollin synthesis in non-elicited tissues. Overexpressing GmJAZ1-9 in hairy roots elicited with Phytophthora sojae wall glucan elicitor partially suppressed glyceollin biosynthesis. The GmJAZ1-9 protein physically interacted with GmNAC42-1 and inhibited its transactivation and DNA binding activities in promoter-luciferase and yeast-three hybrid systems. Silencing JAZ1s in Arabidopsis and grapevine has been reported to derepress camalexin and stilbene phytoalexin biosynthesis. Here, we found that JAZ1 and NAC42 proteins from all three plant species physically interact, suggesting a conserved mechanism negatively regulates phytoalexin biosynthesis in plants. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/615281v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@127cf6borg.highwire.dtl.DTLVardef@a2aa9borg.highwire.dtl.DTLVardef@16ebe08org.highwire.dtl.DTLVardef@17e1cad_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2023

A therapeutic small molecule lead enhances γ-oscillations and improves cognition/memory in Alzheimer's disease model mice

Brain rhythms provide the timing and concurrence of brain activity required for linking together neuronal ensembles engaged in specific tasks. In particular, the {gamma}-oscillations (30-120 Hz) orchestrate neuronal circuits underlying cognitive processes and working memory. These oscillations are reduced in numerous neurological and psychiatric disorders, including early cognitive decline in Alzheimers disease (AD). Here we report on a potent brain permeable small molecule, DDL-920 that increases {gamma}-oscillations and improves cognition/memory in a mouse model of AD, thus showing promise as a new class of therapeutics for AD. As a first in CNS pharmacotherapy, our lead candidate acts as a potent, efficacious, and selective negative allosteric modulator (NAM) of the {gamma}-aminobutyric acid type A receptors (GABAARs) assembled from 1{beta}2{delta} subunits. We identified these receptors through anatomical and pharmacological means to mediate the tonic inhibition of parvalbumin (PV) expressing interneurons (PV+INs) critically involved in the generation of {gamma}-oscillations. Our approach is unique as it is meant to enhance cognitive performance and working memory in a state-dependent manner by engaging and amplifying the brains endogenous {gamma}-oscillations through enhancing the function of PV+INs.

neuroscience↗

bioRxiv2023

Discovery of an APP-Selective BACE1 Inhibitor for Alzheimer's Disease

Inhibition of amyloid precursor protein (APP) beta-site cleaving enzyme 1 (BACE1; BACE) has been a target for Alzheimers disease (AD) therapeutic development, but has been impaired by off-target effects of clinically evaluated inhibitors, including inhibition of cleavage of non-APP substrates. Here, we report our identification of a BACE inhibitors series that are not only selective for the APP substrate, but also for BACE1 as the targeted enzyme. These APP-selective fluoro aminohydantoin (FAH) inhibitor compounds were identified by screening a compound library for inhibition of BACE cleavage of a maltose binding protein (MBP)-conjugated-APPC125 substrate followed by IC50 determination using the P5-P5 substrate assay. In multiple substrate and enzyme cell-free assays, the lead compound FAH65 displayed substrate selectivity for inhibition of APP cleavage, with little activity against BACE substrates neuregulin 1 (NRG1) or p-selectin glycoprotein ligand -1 (PSGL1). We also demonstrate FAH65 shows little inhibitory activity against the enzyme cathepsin D (Cat D) or BACE2. FAH65 inhibits production of BACE cleavage products soluble APP{beta} (sAPP{beta}) and the {beta} C-terminal fragment ({beta}CTF), as well as amyloid-{beta} (A{beta})1-40 and 1-42, in vitro in cells and in vivo in an animal model of AD. In a murine model of AD, FAH65 improved the discrimination score in the Novel Object Recognition (NOR) memory testing paradigm. The active enantiomer of FAH65, FAH65E(-), was obtained and tested in in vivo pharmacokinetic and pharmacodynamic (PK/PD) analysis, wherein it displayed good brain-penetrance and target engagement. Given its demonstrated selectivity for both enzyme and substrate, along with evidence it can improve cognitive performance in an animal model, FAH65 and its E(-) enantiomer merit continued pre-clinical development towards clinical testing as an APP-selective BACE1 inhibitor. Such a candidate would reduce A{beta} levels and overcome the deleterious effects of the non-selective BACE1 inhibitors that have failed in the clinic and potentially could be used as a maintenance therapy along with or following clearance of A{beta} from the brain with the approved antibody therapy for AD.

neuroscience↗