Biology subjects

Nguyen, D. N.

Publications and source records attributed to Nguyen, D. N..

30 records · Page 2Linked to original sources

bioRxiv2023

Altered hepatic metabolism mediates sepsis preventive effects of reduced glucose supply in infected preterm newborns

Preterm infants are susceptible to neonatal sepsis, a syndrome of pro-inflammatory activity, organ damage and altered metabolism following infection. Given the unique metabolic challenges and poor glucose regulatory capacity of preterm infants, their glucose intake during infection may have a high impact on the degree metabolism dysregulation and organ damage. Using a preterm pig model of neonatal sepsis, we previously showed that a drastic restriction in glucose supply during infection protects against sepsis via suppression of glycolysis-induced inflammation, but results in severe hypoglycemia. Now we explored clinically relevant options of reducing glucose intake to decrease sepsis risk, without causing hypoglycemia and further explore the involvement of the liver in these protective effects. We found that a reduced glucose regime during infection increased survival via reduced pro-inflammatory response, while maintaining normoglycemia. Mechanistically, this intervention enhanced hepatic oxidative phosphorylation and possibly gluconeogenesis, and dampened both circulating and hepatic inflammation. However, switching from a high to a reduced glucose supply after debut of clinical symptoms did not prevent sepsis, suggesting metabolic conditions at the start of infection are key in driving the outcome. Finally, an early therapy with purified human inter-alpha inhibitor protein, a liver derived anti-inflammatory protein, partially reversed the effects of low parenteral glucose provision, likely by inhibiting neutrophil functions that mediate pathogen clearance. Our findings suggest a clinically relevant regime of reduced glucose supply for infected preterm infants could prevent or delay the development of sepsis in vulnerable neonates.

immunology↗

bioRxiv2023

Insulin-like Growth Factor-1 Supplementation Promotes Kidney Development and Alleviate Renal Inflammation in Preterm Pigs

BackgroundPreterm birth and its associated complications cause disruption of normal prenatal renal development, leading to postnatal kidney injury and failure. Preterm infants are deficient in insulin-like growth factor 1 (IGF-1), a critical growth factor that stimulates tissue perfusion and development. Using necrotizing enterocolitis-sensitive preterm pigs as a model for preterm infants, we investigated whether IGF-1 supplementation during early life could improve kidney development and health. MethodsCaesarean-delivered preterm pigs were allocated into two groups, either consistently receiving vehicle or IGF-1 immediately after birth for 5, 9 or 19 days. Postnatal age-matched term pigs were selected and served as term control on postnatal day (PND) 5, 9, and 19. Blood, urine and kidney tissue were collected for biochemical, histological and gene expression analyses. ResultsPreterm pigs showed impaired kidney development and increased kidney insults, as indicated by reduced average glomerular area, increased abnormal glomeruli percentage and increased markers of renal injury and inflammation compared to term pigs. IGF-1 supplementation significantly reduced the abnormal glomeruli percentage, renal injury and inflammation related markers, and up-regulated certain maturation-related genes on PND5. ConclusionIGF-1 supplementation supports kidney maturation and restoration of kidney insults after preterm birth in the early life of newborns. ImpactO_LIPreterm birth disrupts kidney development in preterm pigs. C_LIO_LIPreterm birth leads to kidney injury and inflammation in preterm pigs. C_LIO_LIIGF-1 supplementation might promote kidney maturation and alleviate preterm birth associated kidney injury and inflammation in preterm pigs. C_LI

physiology↗

bioRxiv2022

Molecular basis for antiviral activity of pediatric neutralizing antibodies targeting SARS-CoV-2 Spike receptor binding domain

Neutralizing antibodies (NAbs) hold great promise for clinical interventions against SARS-CoV- 2 variants of concern (VOCs). Understanding NAb epitope-dependent antiviral mechanisms is crucial for developing vaccines and therapeutics against VOCs. Here we characterized two potent NAbs, EH3 and EH8, isolated from an unvaccinated pediatric patient with exceptional plasma neutralization activity. EH3 and EH8 cross-neutralize the early VOCs and mediate strong Fc-dependent effector activity in vitro. Structural analyses of EH3 and EH8 in complex with the receptor-binding domain (RBD) revealed the molecular determinants of the epitope-driven protection and VOC-evasion. While EH3 represents the prevalent IGHV3-53 NAb whose epitope substantially overlaps with the ACE2 binding site, EH8 recognizes a narrow epitope exposed in both RBD-up and RBD-down conformations. When tested in vivo, a single-dose prophylactic administration of EH3 fully protected stringent K18-hACE2 mice from lethal challenge with Delta VOC. Our study demonstrates that protective NAbs responses converge in pediatric and adult SARS-CoV-2 patients.

microbiology↗

bioRxiv2021

Engineered ACE2-Fc counters murine lethal SARS-CoV-2 infection through direct neutralization and Fc-effector activities

Soluble Angiotensin-Converting Enzyme 2 (ACE2) constitutes an attractive antiviral capable of targeting a wide range of coronaviruses utilizing ACE2 as their receptor. Here, using structure-guided approaches, we developed divalent ACE2 molecules by grafting the extracellular ACE2-domain onto a human IgG1 or IgG3 (ACE2-Fc). These ACE2-Fcs harbor structurally validated mutations that enhance spike (S) binding and remove angiotensin enzymatic activity. The lead variant bound tightly to S, mediated in vitro neutralization of SARS-CoV-2 variants of concern (VOCs) with sub-nanomolar IC50 and was capable of robust Fc-effector functions, including antibody-dependent-cellular cytotoxicity, phagocytosis and complement deposition. When tested in a stringent K18-hACE2 mouse model, it delayed death or effectively resolved lethal SARS-CoV-2 infection in a prophylactic or therapeutic setting utilizing the combined effect of neutralization and Fc-effector functions. These data confirm the utility of ACE2-Fcs as valuable agents in preventing and eliminating SARS-CoV-2 infection and demonstrate that ACE2-Fc therapeutic activity require Fc-effector functions.

microbiology↗

bioRxiv2021

Parenteral glucose supply and pharmacological glycolysis inhibition determine the clinical fate of infected preterm newborns

Preterm infants are susceptible to bloodstream infection that can lead to sepsis. High parenteral glucose supplement is commonly used to support their growth and energy expenditure, but may exceed endogenous regulation during infection, causing dysregulated immune response and clinical deterioration. Using a preterm piglet model of neonatal sepsis induced by Staphylococcus epidermidis infection, we demonstrate the delicate interplay between immunity and energy metabolism to regulate the host infection response. Circulating glucose levels, glycolysis and inflammatory response to infection are closely connected across the states of tolerance, resistance and immunoparalysis. Further, high parenteral glucose provision during infection induces hyperglycemia, elevated glycolysis and inflammation, leading to lactate acidosis and sepsis, whereas glucose restricted individuals are clinically unaffected with increased gluconeogenesis to maintain moderate hypoglycemia. Finally, pharmacological glycolysis inhibition during normoglycemia enhances bacterial clearance and dampens inflammation but fails to prevent sepsis. Our results uncover how blood glucose controls immune cell metabolism and function, in turn determining the clinical fate of infected preterm neonates. This also questions the current practice of parenteral glucose supply for infected preterm infants.

pathology↗

bioRxiv2021

Hybrid ssDNA repair templates enable high yield genome engineering in primary cells for disease modeling and cell therapy manufacturing

CRISPR-Cas9 offers unprecedented opportunities to modify genome sequences in primary human cells to study disease variants and reprogram cell functions for next-generation cellular therapies. CRISPR has several potential advantages over widely used retroviral vectors including: 1) site-specific transgene insertion via homology directed repair (HDR), and 2) reductions in the cost and complexity of genome modification. Despite rapid progress with ex vivo CRISPR genome engineering, many novel research and clinical applications would be enabled by methods to further improve knock-in efficiency and the absolute yield of live knock-in cells, especially with large HDR templates (HDRT). We recently reported that Cas9 target sequences (CTS) could be introduced into double-stranded DNA (dsDNA) HDRTs to improve knock-in, but yields and efficiencies were limited by toxicity at high HDRT concentrations. Here we developed a novel system that takes advantage of lower toxicity with single-stranded DNA (ssDNA). We designed hybrid ssDNA HDRTs that incorporate CTS sites and were able to boost knock-in percentages by >5-fold and live cell yields by >7-fold relative to dsDNA HDRTs with CTS. Knock-in efficiency and yield with ssCTS HDRTs were increased further with small molecule inhibitor combinations to improve HDR. We demonstrate application of these methods across a variety of target loci, knock-in constructs, and primary human cell types to reach ultra-high HDR efficiencies (>80-90%) which we use for pathogenic gene variant modeling and universal gene replacement strategies for IL2RA and CTLA4 mutations associated with mendelian immune disorders. Finally, we develop a GMP-compatible method for fully non-viral CAR-T cell manufacturing, demonstrating knock-in efficiencies of 46-62% and generating yields of >1.5 x 109 CAR+ T cells, well above current doses for adoptive cellular therapies. Taken together, we present a comprehensive non-viral approach to model disease associated mutations and re-write targeted genome sequences to program immune cell therapies at a scale compatible with future clinical application.

synthetic biology↗

bioRxiv2020

Necrotizing enterocolitis-induced systemic immune suppression in neonatal preterm pigs

ObjectivesPreterm infants are at high risks of sepsis and necrotising enterocolitis (NEC). Some develop sepsis shortly after suspected or confirmed NEC, implying that NEC may predispose to sepsis but the underlying mechanisms are unknown. Using NEC-sensitive preterm pigs as models, we investigated the immune status in animals with and without NEC. MethodsPreterm pigs (n=113, caesarean delivered at day 106) were reared until day 5 or 9. Blood was analyzed for T cell subsets, neutrophil phagocytosis, trans criptomics and immune responses to LPS challenge. Gut tissues were used for histology and cytokine analyses. Pigs with/without macroscopic NEC lesions were scored as healthy, mild or severe NEC. ResultsOverall NEC incidence was similar on days 5 and 9 (61-62%) with less severe lesions on day 9, implying gradual mucosal repair following the early phase of NEC on day 5. Pigs with NEC, especially severe NEC, showed decreased goblet cell density and increased MPO+ and CD3+ cell density in the distal intestine or colon. Circulating parameters were minimally affected by NEC on day 5, but widely altered on day 9 in pigs with NEC, especially severe NEC, to the direction of immune suppression. These included elevated Treg frequency, impaired neutrophil phagocytosis, diminished LPS-induced cytokine secretions and immune gene responses, and consistently low expressions of genes related to innate immune signalling and Th1 polarization. ConclusionWe shows evidence for NEC-induced systemic immune suppression, even with mild and sub-clinical NEC lesions, thereby suggesting mechanisms for increased secondary infections in infants with previous NEC diagnosis.

pathology↗

bioRxiv2020

Fecal filtrate transfer protects against necrotizing enterocolitis in preterm pigs

Background and aimsNecrotizing enterocolitis (NEC) is an acute and life-threatening gastrointestinal disorder afflicting preterm infants, which is currently unpreventable. Fecal microbiota transplantation (FMT) is a promising preventative therapy, but potential side effects raise concern. Removal of bacteria from donor fecal water may reduce side effects while maintaining wanted effects. We aimed to assess preclinical efficacy and safety of bacteria-free fecal filtrate transfer (FFT). MethodsUsing fecal material from healthy suckling piglets, we administered rectal FMT or cognate FFT by either rectal or oro-gastric administration to formula-fed preterm, cesarean piglets, and compared gut pathology and related safety parameters with saline controls. We then analyzed mucosa and luminal bacterial and viral composition using 16S rRNA gene amplicon and metavirome sequencing, respectively. Finally, we used isolated ileal mucosa, coupled with RNA-Seq, to gauge the host response to the different treatments. ResultsOro-gastric FFT eliminated NEC, which was confirmed by microscopy, whereas FMT did not perform better than control. Moreover, FFT but not FMT reduced intestinal permeability, whereas FMT animals had reduced body weight increase and intestinal growth. Oro-gastric FFT increased viral diversity and reduced Proteobacteria abundance in ileal mucosa relative to control. Global gene expression of host mucosa responded to FMT but not FFT with increased and decreased bacterial and viral defense mechanisms, respectively. ConclusionsAs preterm infants are extremely vulnerable, rational therapies need incontestable safety profiles. Here we show in a clinically relevant animal model that FFT, as opposed to FMT, efficiently prevents NEC without any recognizable side effects. If translatable to preterm infants, this could lead to a change of practice and in turn a reduction in NEC burden.

microbiology↗

bioRxiv2019

Rapid discovery of synthetic DNA sequences to rewrite endogenous T cell circuits

Genetically-engineered immune cell therapies have been in development for decades1-3 and recently have proven effective to treat some types of cancer4. CRISPR-based genome editing methods, enabling more flexible and targeted sequence integrations than viral transduction, have the potential to extend the clinical utility of cell therapies5,6. Realization of this potential depends on improved knowledge of how coding and non-coding sites throughout the genome can be modified efficiently and on improved methods to discover novel synthetic DNA sequences that can be introduced at targeted sites to enhance critical immune cell functions. Here, we developed improved guidelines for non-viral genome targeting in human T cells and a pooled discovery platform to identify synthetic genome modifications that enhance therapeutically-relevant cell functions. We demonstrated the breadth of targetable genomic loci by performing large knock-ins at 91 different genomic sites in primary human T cells, and established the power of flexible genome targeting by generating cells with Genetically Engineered Endogenous Proteins (GEEPs) that seamlessly integrate synthetic and endogenous genetic elements to alter signaling input, output, or regulatory control of genes encoding key immune receptors. Motivated by success in introducing synthetic circuits into endogenous sites, we then developed a platform to facilitate discovery of novel multi-gene sequences that reprogram both T cell specificity and function. We knocked in barcoded pools of large DNA sequences encoding polycistronic gene programs. High-throughput pooled screening of targeted knock-ins to the endogenous T cell receptor (TCR) locus revealed a transcriptional regulator and novel protein chimeras that combined with a new TCR specificity to enhance T cell responses in the presence of suppressive conditions in vitro and in vivo. Overall, these pre-clinical studies provide flexible tools to discover complex synthetic gene programs that can be written into targeted genome sites to generate more effective therapeutic cells.

immunology↗

bioRxiv2019

A Cas9 nanoparticle system with truncated Cas9 target sequences on DNA repair templates enhances genome targeting in diverse human immune cell types

Virus-modified T cells are approved for cancer immunotherapy, but more versatile and precise genome modifications are needed for a wider range of adoptive cellular therapies1-4. We recently developed a non-viral CRISPR-Cas9 system for genomic site-specific integration of large DNA sequences in primary human T cells5. Here, we report two key improvements for efficiency and viability in an expanded variety of clinically-relevant primary cell types. We discovered that addition of truncated Cas9 target sequences (tCTS) at the ends of the homology directed repair (HDR) templates can interact with Cas9 ribonucleoproteins (RNPs) to shuttle the template and enhance targeting efficiency. Further, stabilizing the Cas9 RNPs into nanoparticles with poly(glutamic acid) improved editing, reduced toxicity, and enabled lyophilized storage without loss of activity. Combining the tCTS HDR template modifications with polymer-stabilized nanoparticles increased gene targeting efficiency and viable cell yield across multiple genomic loci in diverse cell types. This system is an inexpensive, user-friendly delivery platform for non-viral genome reprogramming that we successfully applied in regulatory T cells (Tregs), {gamma}{delta}-T cells, B cells, NK cells, and primary and iPS-derived6 hematopoietic stem progenitor cells (HSPCs).

bioengineering↗

bioRxiv2018

Timed inhibition of CDC7 increases CRISPR-Cas9 mediated templated repair

Repair of double strand DNA breaks (DSBs) can result in gene disruption or precise gene modification via homology directed repair (HDR) from a templating donor DNA. During genome editing, altering cellular responses to DSBs may be an effective strategy to rebalance editing outcomes towards HDR and away from other repair pathways. To identify factors that regulate HDR from a double-stranded DNA donor (dsDonor), we utilized a pooled screen to define the consequences of thousands of individual gene knockdowns during Cas9-initiated HDR from a double strand plasmid donor. We find that templated dsDonor repair pathways are mostly genetically distinct from single strand donor DNA (ssDonor) repair but share aspects that include dependency upon the Fanconi Anemia (FA) pathway. We also identified several factors whose knockdown increases HDR and thus act as repressors of gene modification. Screening available small molecule inhibitors of these repressors revealed that the cell division cycle 7-related protein kinase (CDC7) inhibitor XL413 increases the efficiency of HDR by 2-3 fold in many contexts, including primary T-cells. XL413 stimulates HDR through cell cycle regulation, inducing an early S-phase cell cycle arrest that, to the best of our knowledge, is uncharacterized for Cas9-induced HDR. We anticipate that XL413 and other such rationally developed inhibitors will be useful tools for boosting the efficiency of gene modification.

molecular biology↗

bioRxiv2018

Double Stranded DNA Damage and Genome Editing Trigger Ribosome Remodeling and Translational Shutdown

DNA damage activates a robust transcriptional stress response, but much less is known about how DNA impacts translation. The advent of genome editing via a Cas9-induced DNA double-strand break has intensified interest in understanding cellular responses to DNA damage. Here we find that DNA double-strand breaks (DSBs) induced by Cas9 or other damaging agents lead to a reduction of core ribosomal proteins, RPS27A and RPL40, and that the loss of these proteins is post-transcriptional and p53-independent. DSBs furthermore lead to the shutdown of translation through phosphorylation of eukaryotic initiation factor 2 alpha, and altering these signals affects genome editing outcomes. This DSB translational response is widespread and precedes the transcriptional response. Our results demonstrate that even a single double-strand break can lead to ribosome remodeling and reduced translational output, and suggest caution in interpreting cellular phenotypes measured immediately after genome editing.

molecular biology↗