Biology subjects

Suzuki, K.

Publications and source records attributed to Suzuki, K..

At least 73 records · Page 4Linked to original sources

bioRxiv2023

Therapeutic immune cell engineering with an mRNA : AAV-Sleeping Beauty composite system

Adoptive cell therapy has shown clinical success in patients with hematological malignancies. Immune cell engineering is critical for production, research, and development of cell therapy; however, current approaches for generation of therapeutic immune cells face various limitations. Here, we establish a composite gene delivery system for the highly efficient engineering of therapeutic immune cells. This system, termed MAJESTIC (mRNA AAV-Sleeping-Beauty Joint Engineering of Stable Therapeutic Immune Cells), combines the merits of mRNA, AAV vector, and transposon into one composite system. In MAJESTIC, the transient mRNA component encodes a transposase that mediates permanent genomic integration of the Sleeping Beauty (SB) transposon, which carries the gene-of-interest and is embedded within the AAV vector. This system can transduce diverse immune cell types with low cellular toxicity and achieve highly efficient and stable therapeutic cargo delivery. Compared with conventional gene delivery systems, such as lentiviral vector, DNA transposon plasmid, or minicircle electroporation, MAJESTIC shows higher cell viability, chimeric antigen receptor (CAR) transgene expression, therapeutic cell yield, as well as prolonged transgene expression. CAR-T cells generated by MAJESTIC are functional and have strong anti-tumor activity in vivo. This system also demonstrates versatility for engineering different cell therapy constructs such as canonical CAR, bi-specific CAR, kill switch CAR, and synthetic TCR; and for CAR delivery into various immune cells, including T cells, natural killer cells, myeloid cells, and induced pluripotent stem cells.

bioengineering↗

bioRxiv2023

CTLA-4 tail fusion enhances CAR-T anti-tumor immunity

Chimeric antigen receptor (CAR) T cells are powerful therapeutics; however, their efficacy is often hindered by critical hurdles. Here, utilizing the endocytic feature of the cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) cytoplasmic tail (CT), we reprogram CAR function and substantially enhance CAR-T efficacy in vivo. CAR-T cells with monomeric, duplex, or triplex CTLA-4 CTs (CCTs) fused to the C-terminus of CAR exhibit a progressive increase in cytotoxicity under repeated stimulation, accompanied by reduced activation and production of pro-inflammatory cytokines. Further characterization reveals that CARs with increasing CCT fusion show a progressively lower surface expression, regulated by their constant endocytosis, recycling and degradation under steady state. The molecular dynamics of reengineered CAR with CCT fusion results in reduced CAR-mediated trogocytosis, loss of tumor antigen, and improved CAR-T survival. CARs with either monomeric (CAR-1CCT) or duplex CCTs (CAR-2CCT) have superior anti-tumor efficacy in a relapsed leukemia model. Single-cell RNA sequencing and flow cytometry analysis reveal that CAR-2CCT cells retain a stronger central memory phenotype and exhibit increased persistence. These findings illuminate a unique strategy for engineering therapeutic T cells and improving CAR-T function through synthetic CCT fusion, which is orthogonal to other cell engineering techniques.

immunology↗

bioRxiv2023

Cas12a/Cpf1 knock-in mice enable efficient multiplexed immune cell engineering

Cas9 transgenic animals have drastically accelerated the discovery of novel immune modulators. But due to its inability to process its own CRISPR RNAs (crRNAs), simultaneous multiplexed gene perturbations using Cas9 remains limited, especially by pseudoviral vectors. Cas12a/Cpf1, however, can process concatenated crRNA arrays for this purpose. Here, we created conditional and constitutive LbCas12a knock-in transgenic mice. With these mice, we demonstrated efficient multiplexed gene editing and surface protein knockdown within individual primary immune cells. We showed genome editing across multiple types of primary immune cells including CD4 and CD8 T cells, B cells, and bone-marrow derived dendritic cells. These transgenic animals, along with the accompanying viral vectors, together provide a versatile toolkit for a broad range of ex vivo and in vivo gene editing applications, including fundamental immunological discovery and immune gene engineering.

genetics↗

bioRxiv2023

Modeling the marmoset brain using embryonic stem cell-derived cerebral assembloids

Studying the non-human primate (NHP) brain is required for the translation of rodent research to humans, but remains a challenge for molecular, cellular, and circuit-level analyses in the NHP brain due to the lack of in vitro NHP brain system. Here, we report an in vitro NHP cerebral model using marmoset (Callithrix jacchus) embryonic stem cell-derived cerebral assembloids (CAs) that recapitulate inhibitory neuron migration and cortical network activity. Cortical organoids (COs) and ganglionic eminence organoids (GEOs) were induced from cjESCs and fused to generate CAs. GEO cells expressing the inhibitory neuron marker LHX6 migrated toward the cortical side of CAs. COs developed their neural activity from a synchronized pattern to an unsynchronized pattern as COs matured. CAs showed mature neural activity with an unsynchronized pattern. The marmoset assembloid system will provide an in vitro platform for the NHP neurobiology and facilitate translation into humans in neuroscience research, regenerative medicine, and drug discovery.

neuroscience↗

bioRxiv2023

Exploratory mass cytometry analysis reveals immunophenotypes of cancer treatment-related pneumonitis

Anti-cancer treatments can result in various adverse effects, including infections due to immune suppression/dysregulation and drug-induced toxicity in the lung. One of the major opportunistic infections is Pneumocystis jirovecii pneumonia (PCP), which can cause severe respiratory complications and high mortality rates. Cytotoxic drugs and immune-checkpoint inhibitors (ICIs) can induce interstitial lung diseases (ILDs). Nonetheless, the differentiation of these diseases can be difficult, and the pathogenic mechanisms of such diseases are not yet fully understood. To better comprehend the immunophenotypes, we conducted an exploratory mass cytometry analysis of immune cell subsets in bronchoalveolar lavage fluid from patients with PCP, cytotoxic drug-induced ILD (DI-ILD), and ICI-associated ILD (ICI-ILD) using two panels containing 64 markers. In PCP, we observed an expansion of the CD16+ T cell population, with the highest CD16+ T proportion in a fatal case. In ICI-ILD, we found an increase in CD57+ CD8+ T cells expressing immune checkpoints (TIGIT+ LAG3+ TIM-3+ PD-1+), FCRL5+ B cells, and CCR2+ CCR5+ CD14+ monocytes. These findings uncover the diverse immunophenotypes and possible pathomechanisms of cancer treatment-related pneumonitis.

immunology↗

bioRxiv2023

Modelling Phenomenological Differences in Aetiologically Distinct Visual Hallucinations Using Deep Neural Networks

Visual hallucinations (VHs) are perceptions of objects or events in the absence of the sensory stimulation that would normally support such perceptions. Although all VHs share this core characteristic, there are substantial phenomenological differences between VHs that have different aetiologies, such as those arising from neurological conditions, visual loss, or psychedelic compounds. Here, we examine the potential mechanistic basis of these differences by leveraging recent advances in visualising the learned representations of a coupled classifier and generative deep neural network - an approach we call computational (neuro)phenomenology. Examining three aetiologically distinct populations in which VHs occur - neurological conditions (Parkinsons Disease and Lewy Body Dementia), visual loss (Charles Bonnet Syndrome, CBS), and psychedelics - we identify three dimensions relevant to distinguishing these classes of VHs: realism (veridicality), dependence on sensory input (spontaneity), and complexity. By selectively tuning the parameters of the visualisation algorithm to reflect influence along each of these phenomenological dimensions we were able to generate synthetic VHs that were characteristic of the VHs experienced by each aetiology. We verified the validity of this approach experimentally in two studies that examined the phenomenology of VHs in neurological and CBS patients, and in people with recent psychedelic experience. These studies confirmed the existence of phenomenological differences across these three dimensions between groups, and crucially, found that the appropriate synthetic VHs were representative of each groups hallucinatory phenomenology. Together, our findings highlight the phenomenological diversity of VHs associated with distinct causal factors and demonstrate how a neural network model of visual phenomenology can successfully capture the distinctive visual characteristics of hallucinatory experience.

neuroscience↗

bioRxiv2023

Facilitative interaction networks in experimental microbial community dynamics

Understanding potential roles of facilitative interactions between species is one of the major challenges in ecology and microbiology. However, we still have limited knowledge of entangled webs of facilitative interactions in ecosystems. By compiling whole-genome shotgun metagenomic data of an experimental microbial community, we tested the hypothesis that architecture of facilitative interaction networks could change through time. A metabolic modeling approach for estimating dependence between microbial genomes (species) allowed us to infer the network structure of potential facilitative interactions at 13 time points through the 110-day monitoring of experimental microbiomes. We then found that positive feedback loops, which were theoretically predicted to promote cascade breakdown of ecological communities, existed within the inferred networks of metabolic interactions prior to the drastic community-compositional shift observed in the microbiome time-series. We further applied "directed-graph" analyses to pinpoint potential keystone species located at the "upper stream" positions of such feedback loops. These analyses on facilitative interactions will help us understand key mechanisms causing catastrophic shifts in microbial community structure.

ecology↗

bioRxiv2023

Metagenomic analysis of ecological niche overlap and community collapse in microbiome dynamics

Species utilizing the same resources often fail to coexist for extended periods of time. Such competitive exclusion mechanisms potentially underly microbiome dynamics, causing breakdowns of communities composed of species with similar genetic backgrounds of resource utilization. Although genes responsible for competitive exclusion among a small number of species have been investigated in pioneering studies, it remains a major challenge to integrate genomics and ecology for understanding stable coexistence in species-rich communities. Here, we show that community-scale analyses of functional gene redundancy can provide a useful platform for interpreting and predicting collapse of bacterial communities. Through 110-day time-series of experimental microbiome dynamics, we analyzed the metagenome-assembled genomes of co-occurring bacterial species. We then inferred ecological niche space based on the multivariate analysis of the genome compositions. The analysis allowed us to evaluate potential shifts in the level of niche overlap between species through time. We hypothesized that community-scale pressure of competitive exclusion could be evaluated by quantifying overlap of genetically determined resource-use profiles (metabolic pathway profiles) among coexisting species. We found that the degree of community compositional changes observed in the experimental microbiome was explained by the magnitude of gene-repertoire overlaps among bacterial species. The metagenome-based analysis of genetic potential for competitive exclusion will help us forecast major events in microbiome dynamics such as sudden community collapse (i.e., dysbiosis).

microbiology↗

bioRxiv2023

Mass cytometry identifies characteristic immune cell subsets in bronchoalveolar lavage fluid from interstitial lung diseases

Immune cells have been implicated in interstitial lung diseases (ILDs), although their phenotypes and effector mechanisms remain poorly understood. To better understand these cells, we conducted an exploratory mass cytometry analysis of immune cell subsets in bronchoalveolar lavage fluid (BALF) from patients with idiopathic pulmonary fibrosis (IPF), connective-tissue disease (CTD)-related ILD, and sarcoidosis, using two panels including 64 markers. Among myeloid cells, we observed the expansion of CD14+ CD36hi CD84hi monocyte populations in IPF. These CD14+ CD36hi CD84hi subsets were also increased in ILDs with a progressive phenotype, particularly in a case of acute exacerbation (AEx) of IPF. Analysis of B cells revealed the presence of cells at various stages of differentiation in BALF, with a higher percentage of IgG memory B cells in CTD-ILDs and a trend toward more FCRL5+ B cells. These FCRL5+B cells were also present in the patient with AEx-IPF and sarcoidosis with advanced lung lesions. Among T cells, we found increased levels of IL-2R+TIGIT+ LAG3+ CD4+T cells in IPF, increased levels of CXCR3+ CD226+ CD4+ T cells in sarcoidosis, and increased levels of PD1+ TIGIT+ CD57+ CD8+ T cells in CTD-ILDs. Together, these findings underscore the diverse immunopathogenesis of ILDs.

immunology↗

bioRxiv2022

Polyvalent mRNA vaccination elicited potent immune response to monkeypox surface antigens

The soaring global monkeypox cases lead to a surge in demand for monkeypox vaccine, which far exceeds the supply. mRNA vaccine has achieved great success in prevention of coronavirus disease and holds promise against diverse pathogens. In this study, we generate a polyvalent lipid nanoparticle (LNP) mRNA vaccine candidate for monkeypox virus (MPXV) and evaluate its immunogenicity in animal models. This polyvalent MPXV mRNA vaccine candidate, MPXVac-097, encodes five 2022 MPXV targets that are important surface antigens. Three-dose (prime-boost-booster) MPXVac-097 vaccination elicits strong antibody response to A35R and E8L antigens, moderate response to M1R, but not B6R or A29, highlighting the differences in immunogenicity. Bulk T cell receptor (TCR) sequencing reveals preferential usage of VJ combinations and clonal expansion of peripheral T cells after MPXVac-097 vaccination. These data demonstrate initial feasibility of developing MPXV mRNA vaccine and pave the way for its future optimization.

immunology↗

bioRxiv2022

Immune Isoform Atlas: Landscape of alternative splicing in human immune cells

Alternative splicing events are a major causal mechanism for complex traits, but they have been understudied due to the limitation of short-read sequencing. Here, we generated a comprehensive full-length isoform annotation of human immune cells, Immune Isoform Atlas, by long-read sequencing for 29 cell subsets. Our atlas contained a number of unannotated transcripts and isoforms such as a read-through transcript of TOMM40-APOE. We profiled functional characteristics of isoforms including encoded domains, inserted repetitive elements, and translational efficiency, and we showed that repetitive elements significantly explained the diversity of unannotated isoforms. Some of the isoforms are expressed in a cell-type specific manner, whose alternative 3-UTRs usage contributed to their specificity. Further, we identified a number of disease-associated isoforms by isoform switch analysis and by integration of several quantitative trait loci analyses with genome-wide association study data. Our findings will promote the elucidation of the pathomechanism of diseases via alternative splicing.

genetics↗

bioRxiv2022

Alternative stable states, nonlinear behavior, and predictability of microbiome dynamics

Microbiome dynamics are both crucial indicators and drivers of human health, agricultural output, and industrial bio-applications. However, predicting microbiome dynamics is notoriously difficult because communities often show abrupt structural changes, such as "dysbiosis" in human microbiomes. We here integrate theoretical and empirical bases for anticipating drastic shifts of microbial communities. We monitored 48 experimental microbiomes for 110 days and observed that various community-level events, including collapse and gradual compositional changes, occurred according to a defined set of environmental conditions. We then confirmed that the abrupt community changes observed through the time-series could be described as shifts between "alternative stable states" or dynamics around complex attractors. Furthermore, collapses of microbiome structure were successfully anticipated by means of the diagnostic threshold defined with the energy landscape analysis of statistical physics or that of a stability index of nonlinear mechanics. These results indicate that abrupt microbiome events in complex microbial communities can be forecasted by extending classic ecological concepts to the scale of species-rich microbial systems.

ecology↗

bioRxiv2022

Stable states and keystone species in soil microbiomes of agroecosystems

Theory predicts that biological communities can have multiple basins of attraction in terms of their species/taxonomic compositions. The presence of such basins of community structure has been examined in classic empirical studies on forest-savanna transitions and those on eutrophication in freshwater lakes. Nonetheless, it remains a major challenge to extend the investigations of multistability to species-rich microbial communities. By targeting soil microbiomes, we infer the stability landscapes of community structure based on the concepts of statistical physics. Our analysis on the compiled dataset involving 11 archaeal, 332 bacterial, and 240 fungal families detected from > 1,500 agroecosystem soil samples suggested that both prokaryotic and fungal community compositions could be classified into several basins of attraction. We also found that the basins differed greatly in their associations with crop disease prevalence in agroecosystems. A further analysis highlighted microbial taxa potentially playing key roles in transitions between basins with different ecosystem-scale functions. The statistical framework commonly applicable to diverse microbial and non-microbial communities will reorganize our understanding of relationship among community structure, stability, and functions.

microbiology↗

bioRxiv2022

Function and Cryo-EM structures of broadly potent bispecific antibodies against multiple SARS-CoV-2 Omicron sublineages

The SARS-CoV-2 variant, Omicron (B.1.1.529), rapidly swept the world since its emergence. Compared with previous variants, Omicron has a high number of mutations, especially those in its spike glycoprotein that drastically dampen or abolish the efficacy of currently available vaccines and therapeutic antibodies. Several major sublineages of Omicron evolved, including BA.1, BA.1.1, BA.2, BA.2.12.1, BA.3, BA.4/5, and BA.2.75, which rapidly changing the global and regional landscape of the pandemic. Although vaccines are available, therapeutic antibodies remain critical for infected and especially hospitalized patients. To address this, we have designed and generated a panel of human/humanized therapeutic bispecific antibodies against Omicron and its sub-lineage variants, with activity spectrum against other lineages. Among these, the top clone CoV2-0213 has broadly potent activities against multiple SARS-CoV-2 ancestral and Omicron lineages, including BA.1, BA.1.1, BA.2, BA.2.12.1, BA.3, BA.4/5, and BA.2.75. We have solved the cryo-EM structure of the lead bi-specific antibody CoV-0213 and its major Fab arm MB.02. Three-dimensional structural analysis shows distinct epitope of antibody - spike receptor binding domain (RBD) interactions and reveals that both Fab fragments of CoV2-0213 can simultaneously target one single spike RBD or two adjacent ones in the same spike trimer, further corroborating its mechanism of action. CoV2-0213 represents a unique and potent broad-spectrum SARS-CoV-2 neutralizing bispecific antibody (nbsAb) against the currently circulating major Omicron variants (BA.1, BA.1.1, BA.2, BA.2.12.1, BA.2.75, BA.3, and BA.4/5). CoV2-0213 is primarily human and ready for translational testing as a countermeasure against the ever-evolving pathogen.

immunology↗

bioRxiv2022

Fc-modified SARS-CoV-2 neutralizing antibodies with therapeutic effects in two animal models.

The use of therapeutic neutralizing antibodies against SARS-CoV-2 infection has been highly effective. However, there remain few practical antibodies against viruses that are acquiring mutations. In this study, we created 494 monoclonal antibodies from COVID-19-convalescent patients, and identified antibodies that exhibited comparable neutralizing ability to clinically used antibodies in the neutralization assay using pseudovirus and authentic virus including variants of concerns. These antibodies have different profiles against various mutations, which were confirmed by cell-based assay and cryo-electron microscopy. To prevent antibody-dependent enhancement, N297A modification was introduced, and showed a reduction of lung viral RNAs by therapeutic administration in a hamster model. In addition, an antibody cocktail consisting of three antibodies was also administered therapeutically to a macaque model, which resulted in reduced viral titers of swabs and lungs and reduced lung tissue damage scores. These results showed that our antibodies have sufficient antiviral activity as therapeutic candidates.

microbiology↗

bioRxiv2022

Reprogramming of three-dimensional microenvironments for in vitro hair follicle induction

During embryonic development, reciprocal interactions between epidermal and mesenchymal layers trigger hair follicle morphogenesis. This study revealed that microenvironmental reprogramming via control over these interactions enabled hair follicle induction in vitro. A key approach is to modulate spatial distributions of epithelial and mesenchymal cells in their spontaneous organization. The de novo hair follicles with typical morphological features emerged in aggregates of the two cell types, termed hair follicloids, and hair shafts sprouted with near 100% efficiency in vitro. The hair shaft length reached [~]3 mm in culture. Typical trichogenic signaling pathways were upregulated in hair follicloids. Owing to replication of hair follicle morphogenesis in vitro, production and transportation of melanosomes were also monitored in the hair bulb region. This in vitro hair follicle model might be valuable for better understanding hair follicle induction, for evaluating hair growth as well as the inhibition of hair growth by drugs, and modeling gray hairs in a well-defined environment. TeaserIn tissue morphogenesis, different types of cells harmonize in a pre-programmed manner using messenger systems such as epithelial-mesenchymal interactions. Organoids are a promising tool to elucidate such mechanisms on a molecular level. This work describes a strategy for reprograming three-dimensional microenvironments to trigger the initiation of in vitro regeneration of hair follicle organoids. Hair follicle organoids generated fully matured hair follicles, enabling the monitoring of hair follicle morphogenesis in vitro and determination of signaling pathways involved in early hair follicle morphogenesis. The principles uncovered herein may be relevant to other organ systems and will contribute to our understanding of developmental phenomena in physiological and pathological processes, eventually opening up new research avenues for the development of new treatment strategies.

bioengineering↗

bioRxiv2022

Prime editing in chicken fibroblasts and primordial germ cells

CRISPR/Cas9-based genome editing technologies are revolutionizing developmental biology. One of the advanced CRISPR-based techniques is prime editing (PE), which enables precise gene modification in multiple model organisms. In the current study, we describe a method to apply PE to the genome of chicken fibroblasts and primordial germ cells (PGCs). By combining PE with a transposon-mediated genomic integration, drug selection, and the single-cell culture method, we successfully generated prime-edited chick PGCs. The chicken PGC is widely used as an experimental model to study germ cell formation and as a vector for gene transfer to produce transgenic chickens. Such experimental models are useful in the developmental biology field and as potential bioreactors to produce pharmaceutical and nutritious proteins. Thus, the method presented here will provide not only a powerful tool to investigate gene function in germ cell development but also a basis for generating prime-edited transgenic birds.

developmental biology↗

bioRxiv2022

Redox sensing by SENP1 augments insulin secretion early after high-fat feeding in mice

Pancreatic {beta}-cells respond to metabolic stress by upregulating insulin secretion, however the underlying mechanisms remain unclear. In {beta}-cells from overweight humans without diabetes, and mice fed a high-fat diet for 2 days, insulin exocytosis and secretion are enhanced without increased Ca2+ influx. {beta}-cell RNA-seq suggests altered metabolic pathways early following HFD, where we find increased basal oxygen consumption, proton leak, but a more reduced cytosolic redox state. Increased {beta}-cell exocytosis after 2-day HFD is dependent on this reduced intracellular redox and requires the sentrin-specific SUMO-protease-1 (SENP1). Mice with either pancreas- or {beta}-cell-specific SENP1 deletion fail to up-regulate exocytosis and become rapidly glucose intolerant after 2-day HFD. Mechanistically, redox-sensing by SENP1 requires a thiol group at C535 which together with Zn+-binding suppresses basal protease activity and unrestrained {beta}-cell exocytosis and increases SENP1 sensitivity to regulation by redox signals.

cell biology↗