技术资料

Genome editing using CRISPR-Cas systems is a promising avenue for the treatment of genetic diseases. However,cellular and humoral immunogenicity of genome editing tools,which originate from bacteria,complicates their clinical use. Here we report reduced immunogenicity (Red)(i)-variants of two clinically relevant nucleases,SaCas9 and AsCas12a. Through MHC-associated peptide proteomics (MAPPs) analysis,we identify putative immunogenic epitopes on each nuclease. Using computational modeling,we rationally design these proteins to evade the immune response. SaCas9 and AsCas12a Redi variants are substantially less recognized by adaptive immune components,including reduced binding affinity to MHC molecules and attenuated generation of cytotoxic T cell responses,yet maintain wild-type levels of activity and specificity. In vivo editing of PCSK9 with SaCas9.Redi.1 is comparable in efficiency to wild-type SaCas9,but significantly reduces undesired immune responses. This demonstrates the utility of this approach in engineering proteins to evade immune detection. Subject terms: Protein design,Immunogenetics,CRISPR-Cas9 genome editing View Publication

Human norovirus (HuNoV) is a major cause of enteric infectious gastroenteritis and is classified into several genotypes based on its capsid protein amino acid sequence and nucleotide sequence of the polymerase gene. Among these,GII.4 is the major genotype worldwide. Epidemiological studies have highlighted the prevalence of GII.2. Although recent advances using human tissue– and induced pluripotent stem cell (iPSC)–derived intestinal epithelial cells (IECs) have enabled in vitro replication of multiple HuNoV genotypes,GII.2 HuNoV could replicate only in tissue-derived IECs and not in iPSC-derived IECs. We investigated the factors influencing GII.2 HuNoV replication in IECs,focusing on histo-blood group antigens. We also assessed the immunogenicity of GII.2 virus-like particles (VLPs) and their ability to induce neutralizing antibodies. Antibody cross-reactivity was tested to determine whether GII.2 VLPs could neutralize other HuNoV genotypes,including GII.4,GII.3,GII.6,and GII.17. Our findings indicated that GII.2 HuNoV replication in vitro requires the presence of both H and B antigens. Moreover,GII.2 VLPs generated neutralizing antibodies effective against both GII.2 and GII.4 but not against GII.3,GII.6,or GII.17. Comparatively,GII.2 and GII.17 VLPs induced broader neutralizing responses than GII.4 VLPs. The findings of this study suggests that GII.2 and GII.17 VLPs may be advantageous as HuNoV vaccine candidates because they elicit neutralizing antibodies against the predominant GII.4 genotype,which could be particularly beneficial for infants without prior HuNoV exposure. These insights will contribute to the development of effective HuNoV vaccines. View Publication

Polo-like kinase 1 (PLK1),a key regulator of the G2/M phase in mitosis,is frequently overexpressed in numerous tumors. Although PLK1 inhibitors have emerged as promising therapeutic agents for cancer,their use has been linked to significant anemia in a subset of patients,yet the underlying mechanisms remain poorly understood. In this study,we utilized an in vitro human umbilical cord blood-derived CD34 + cell-based erythroid differentiation system,alongside a murine model,to investigate the impact of PLK1 inhibitors on erythropoiesis. Our results indicate that PLK1 inhibitors,specifically GSK461364 and BI6727,significantly suppress the proliferation of erythroid cells,resulting in G2/M phase cell cycle arrest,increased apoptosis in erythroid cells,and the formation of abnormally nucleated late-stage erythroblasts. In vivo,administration of PLK1 inhibitors in mice induced severe anemia,as evidenced by a marked reduction in red blood cells and hemoglobin levels. More specifically,PLK1 inhibition impaired the differentiation and erythroid commitment of hematopoietic stem cells in the bone marrow,resulting in abnormal accumulation of BFU-E cells and reduced proliferation and differentiation of CFU-E,and a decrease in the number of terminal erythrocytes. Mechanistically,PLK1 inhibitors primarily induce apoptosis in erythroid cells by reducing Mitochondrial membrane potential and arresting the cell cycle at the G2/M phase. Overall,our findings underscore the critical role of PLK1 in erythropoiesis and shed light on the mechanisms underlying PLK1 inhibitor-induced anemia,providing essential guidance for developing strategies to prevent and manage anemia in clinical applications of PLK1-targeted therapies. View Publication

Previous studies have indicated that the presence of cancer stem cells may be a contributing factor to the development of metastasis in colorectal cancer patients. Cancer stem cells represent a small subpopulation within the tumor mass that exhibits heightened resistance to treatment and possesses the capacity for self-replication,epithelial–mesenchymal transition,and the generation of new tumors. The tumor microenvironment secretes and releases several molecules that facilitate the self-renewal of cancer stem cells and provide support for colorectal cancer progression. microRNAs are involved in direct cell-to-cell signaling and paracrine signaling between tumor cells and other tumor microenvironment components. They could act as tumor suppressors or oncomiRs,and their deregulation is involved in colorectal cancer progression and cancer stem cell formation. In our previous studies,we demonstrated the oncosuppressive function of miR-486-5p in colorectal cancer; these findings prompted us to conduct a more detailed investigation into its role in cancer stem cell phenotypes. Background: Colorectal cancer (CRC) is the third diagnosed cancer worldwide. Forty-four percent of metastatic colorectal cancer patients were diagnosed at an early stage. Despite curative resection,approximately 40% of patients will develop metastases within a few years. Previous studies indicate the presence of cancer stem cells (CSCs) and their contribution to CRC progression and metastasis. miRNAs deregulation plays a role in CSCs formation and in tumor development. In light of previous studies,we investigated the role of miR-486-5p to understand its role in CSC better. Methods: The expression of miR-486-5p was assessed in adherent cells and spheres generated from two CRC cell lines to observe the difference in expression in CSC-enriched spheroids. Afterward,we overexpressed and underexpressed this miRNA in adherent and sphere cultures through the transfection of a miR-486-5p mimic and a mimic inhibitor. Results: The results demonstrated that miR-486-5p exhibited a notable downregulation in CSC models,and its overexpression led to a significant decrease in colony size. Conclusions: In this study,we confirmed that miR-486-5p plays an oncosuppressive role in CRC,thereby advancing our understanding of the role of this microRNA in the CSC phenotype. View Publication

Cancer stem cells (CSCs) play a key role in non-small cell lung cancer (NSCLC) chemoresistance and metastasis. In this study,we used two NSCLC cell lines to investigate the regulating effect of hypoxia in the induction and maintenance of CSC traits. Our study demonstrated hypoxia-induced stemness and chemoresistance at levels comparable to those in typical CSC sphere culture. Activation of the NF-κB pathway (by transfection of NF-κB-p65) plays a key role in NSCLC CSCs and chemoresistance. Disulfiram (DS),an anti-alcoholism drug,showed a strong in vitro anti-CSC effect. It blocked cancer cell sphere reformation and clonogenicity,synergistically enhanced the cytotoxicity of four anti-NSCLC drugs (doxorubicin,gemcitabine,oxaliplatin and paclitaxel) and reversed hypoxia-induced resistance. The effect of DS on CSCs is copper-dependent. A very short half-life in the bloodstream is the major limitation for the translation of DS into a cancer treatment. Our team previously developed a poly lactic-co-glycolic acid (PLGA) nanoparticle encapsulated DS (DS-PLGA) with a long half-life in the bloodstream. Intra venous injection of DS-PLGA in combination with the oral application of copper gluconate has strong anticancer efficacy in a metastatic NSCLC mouse model. Further study may be able to translate DS-PLGA into cancer applications. View Publication

Melanoma brain metastasis is linked to dismal prognosis and low overall survival and is detected in up to 80% of patients at autopsy. Circulating tumor cells (CTC) are the smallest functional units of cancer and precursors of fatal metastasis. We previously used an unbiased multilevel approach to discover a unique ribosomal protein large/small subunit (RPL/RPS) CTC gene signature associated with melanoma brain metastasis. In this study,we hypothesized that CTC-driven melanoma brain metastasis secondary metastasis (“metastasis of metastasis” per clinical scenarios) has targeted organ specificity for the liver. We injected parallel cohorts of immunodeficient and newly developed humanized NBSGW (huNBSGW) mice with cells from CTC-derived melanoma brain metastasis to identify secondary metastatic patterns. We found the presence of a melanoma brain–liver metastasis axis in huNBSGW mice. Furthermore,RNA sequencing analysis of tissues showed a significant upregulation of the RPL/RPS CTC gene signature linked to metastatic spread to the liver. Additional RNA sequencing of CTCs from huNBSGW blood revealed extensive CTC clustering with human B cells in these mice. CTC:B-cell clusters were also upregulated in the blood of patients with primary melanoma and maintained either in CTC-driven melanoma brain metastasis or melanoma brain metastasis CTC–derived cells promoting liver metastasis. CTC-generated tumor tissues were interrogated at single-cell gene and protein expression levels (10x Genomics Xenium and HALO spatial biology platforms,respectively). Collectively,our findings suggest that heterotypic CTC:B-cell interactions can be critical at multiple stages of metastasis. This study provides important insights into the relevance of prometastatic CTC:B-cell clusters in melanoma progression,extends the importance of the CTC RPL/RPS gene signature beyond primary metastasis/melanoma brain metastasis driving targeted organ specificity for liver metastasis (“metastasis of metastasis”),and identifies new targets for clinical melanoma metastasis therapies. View Publication

Dishevelled-associated activator of morphogenesis1 (DAAM1) is a member of the evolutionarily conserved Formin family and plays a significant role in the malignant progression of various human cancers. This study aims to explore the clinical and biological significance of DAAM1 in pancreatic cancer. Multiple public datasets and an in-house cohort were utilized to assess the clinical relevance of DAAM1 in pancreatic cancer. The LinkedOmics platform was employed to perform enrichment analysis of DAAM1-associated molecular pathways in pancreatic cancer. Subsequently,a series of in vitro and in vivo experiments were conducted to evaluate the biological roles of DAAM1 in pancreatic cancer cells and its effects on intratumoral T cells. DAAM1 was found to be upregulated in pancreatic cancer tissues,with higher expression levels observed in tumor cells. Additionally,high expression of DAAM1 was associated with poor prognosis. DAAM1 acted as an oncogene in pancreatic cancer,and its inhibition suppressed tumor cell proliferation,migration,and invasion,while promoted apoptosis. Furthermore,DAAM1 was involved in the JAK1/STAT1 signaling pathway and regulated PD-L1 expression in pancreatic cancer cells. The inhibition of DAAM1 also significantly reduced the exhaustion levels of CD8+ T cells. In conclusion,DAAM1 functions as an oncogene and is immunologically implicated in pancreatic cancer,these findings suggest that DAAM1 may serve as a promising therapeutic target for the clinical management of pancreatic cancer. The online version contains supplementary material available at 10.1186/s12935-024-03631-8. View Publication

Chimeric antigen receptor (CAR)-engineered T cell therapy holds promise for treating myeloid malignancies,but challenges remain in bone marrow (BM) infiltration and targeting BM-resident malignant cells. Current autologous CAR-T therapies also face manufacturing and patient selection issues,underscoring the need for off-the-shelf products. In this study,we characterize primary patient samples and identify a unique therapeutic opportunity for CAR-engineered invariant natural killer T (CAR-NKT) cells. Using stem cell gene engineering and a clinically guided culture method,we generate allogeneic CD33-directed CAR-NKT cells with high yield,purity,and robustness. In preclinical mouse models,CAR-NKT cells exhibit strong BM homing and effectively target BM-resident malignant blast cells,including CD33-low/negative leukemia stem and progenitor cells. Furthermore,CAR-NKT cells synergize with hypomethylating agents,enhancing tumor-killing efficacy. These cells also show minimal off-tumor toxicity,reduced graft-versus-host disease and cytokine release syndrome risks,and resistance to allorejection,highlighting their substantial therapeutic potential for treating myeloid malignancies. Subject terms: Cancer therapy,Immunotherapy,Leukaemia View Publication

To fully utilize the potential of human induced pluripotent stem cells (hiPSCs) for allogeneic stem cell–based therapies,efficient and scalable expansion procedures must be developed. For other adherent human cell types,the combination of microcarriers (MCs) and stirred tank bioreactors has been shown to meet these demands. In this study,a hiPSC quasi-perfusion expansion procedure based on MCs was developed at 100-mL scale in spinner flasks. Process development began by assessing various medium exchange strategies and MC coatings,indicating that the hiPSCs tolerated the gradual exchange of medium well when cultivated on Synthemax II–coated MCs. This procedure was therefore scaled-up to the 1.3-L Eppendorf BioBLU 1c stirred tank bioreactor by applying the lower limit of Zwietering’s suspension criterion ( N s 1 u ),thereby demonstrating proof-of-concept when used in combination with hiPSCs for the first time. To better understand the bioreactor and its bioengineering characteristics,computational fluid dynamics and bioengineering investigations were performed prior to hiPSC cultivation. In this manner,improved process understanding allowed an expansion factor of ≈ 26 to be achieved,yielding more than 3 × 10 9 cells within 5 days. Further quality analyses confirmed that the hiPSCs maintained their viability,identity,and differentiation potential throughout cultivation. • N s 1 u can be used as a scale-up criterion for hiPSC cultivations in MC-operated stirred bioreactors • Uniform distribution and attachment of cells to the MCs are crucial for efficient expansion • Perfusion is advantageous and supports the cultivation of hiPSCs The online version contains supplementary material available at 10.1007/s00253-024-13372-3. View Publication

Acute myeloid leukemia (AML) is characterized by the accumulation of immature myeloid cells and a differentiation block,highlighting the urgent need for novel differentiation-inducing therapies. This study evaluated Adina rubella Hance (ARH) stem as a potent differentiation inducer by systematically screening 200 plant extracts. ARH stem promoted phenotypic differentiation in AML cells. In addition to its differentiation-inducing effects,ARH stem exhibited strong antileukemic activities,such as inhibiting cell proliferation,inducing cell death,and enhancing mitochondrial reactive oxygen species (mtROS) levels,the latter of which is critical for its differentiation-promoting activity. Comparative analysis with the extracts from other parts of the plant confirmed the superior efficacy of the stem extract because of its unique chemical composition. Ultra-high-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry analysis identified Picroside III as a major active compound within the stem extract,capable of recapitulating ARH stem-induced differentiation and demonstrating significant antileukemic properties. These findings underscore the therapeutic potential of ARH stem and its active component,Picroside III,as promising agents for differentiation-based treatment strategies in AML. View Publication

Understanding the interaction between genetic and epigenetic variation remains a challenge due to confounding environmental factors. We propose that human induced Pluripotent Stem Cells (iPSCs) are an excellent model to study the relationship between genetic and epigenetic variation while controlling for environmental factors. In this study,we have created a comprehensive resource of high-quality genomic,epigenomic,and transcriptomic data from iPSC lines and three iPSC-derived cell types (neural stem cell (NSC),motor neuron,monocyte) from three healthy donors. We find that epigenetic variation is most strongly associated with genetic variation at the iPSC stage,and that relationship weakens as epigenetic variation increases in differentiated cells. Additionally,cell type is a stronger source of epigenetic variation than genetic variation. Further,we elucidate a utility of studying epigenetic variation in iPSCs and their derivatives for identifying important loci for GWAS studies and the cell types in which they may be acting. Subject terms: Epigenomics,Genomics,Transcriptomics View Publication

Persistence of drug-resistant breast cancer stem cells (brCSCs) after a chemotherapeutic regime correlates with disease recurrence and elevated mortality. Therefore,deciphering mechanisms that dictate their drug-resistant phenotype is imperative for designing targeted and more effective therapeutic strategies. The transcription factor SOX2 has been recognized as a protagonist in brCSC maintenance,and previous studies have confirmed that inhibition of SOX2 purportedly eliminated these brCSCs. However,pharmacological targeting of transcription factors like SOX2 is challenging due to their structural incongruities and intrinsic disorders in their binding interfaces. Therefore,transcriptional co-activators may serve as a feasible alternative for effectively targeting the brCSCs. Incidentally,transcriptional co-activators YAP/TAZ were found to be upregulated in CD44 + /CD24 - /ALDH + cells isolated from patient breast tumors and CSC-enriched mammospheres. Interestingly,it was observed that YAP/TAZ exhibited direct physical interaction with SOX2 and silencing YAP/TAZ attenuated SOX2 expression in mammospheres,leading to significantly reduced sphere forming efficiency and cell viability. YAP/TAZ additionally manipulated redox homeostasis and regulated mitochondrial dynamics by restraining the expression of the mitochondrial fission marker,DRP1. Furthermore,YAP/TAZ inhibition induced DRP1 expression and impaired OXPHOS,consequently inducing apoptosis in mammospheres. In order to enhance clinical relevance of the study,an FDA-approved drug verteporfin (VP),was used for pharmacological inhibition of YAP/TAZ. Surprisingly,VP administration was found to reduce tumor-initiating capacity of the mammospheres,concomitant with disrupted mitochondrial homeostasis and significantly reduced brCSC population. Therefore,VP holds immense potential for repurposing and decisively eliminating the chemoresistant brCSCs,offering a potent strategy for managing tumor recurrence effectively. Subject terms: Cancer stem cells,Cancer stem cells View Publication