技术资料

Allogeneic cell therapies,such as those involving macrophages or Natural Killer (NK) cells,are of increasing interest for cancer immunotherapy. However,the current techniques for genetically modifying these cell types using lenti- or gamma-retroviral vectors present challenges,such as required cell pre-activation and inefficiency in transduction,which hinder the assessment of preclinical efficacy and clinical translation. In our study,we describe a novel lentiviral pseudotype based on the Koala Retrovirus (KoRV) envelope protein,which we identified based on homology to existing pseudotypes used in cell therapy. Unlike other pseudotyped viral vectors,this KoRV-based envelope demonstrates remarkable efficiency in transducing freshly isolated primary human NK cells directly from blood,as well as freshly obtained monocytes,which were differentiated to M1 macrophages as well as B cells from multiple donors,achieving up to 80% reporter gene expression within three days post-transduction. Importantly,KoRV-based transduction does not compromise the expression of crucial immune cell receptors,nor does it impair immune cell functionality,including NK cell viability,proliferation,cytotoxicity as well as phagocytosis of differentiated macrophages. Preserving immune cell functionality is pivotal for the success of cell-based therapeutics in treating various malignancies. By achieving high transduction rates of freshly isolated immune cells before expansion,our approach enables a streamlined and cost-effective automated production of off-the-shelf cell therapeutics,requiring fewer viral particles and less manufacturing steps. This breakthrough holds the potential to significantly reduce the time and resources required for producing e.g. NK cell therapeutics,expediting their availability to patients in need. Subject terms: Genetic transduction,Tumour immunology,Immunotherapy,Genetic vectors,Innate immune cells View Publication

In preclinical studies on the T-cell engager MP0533,the authors show that targeting multiple tumor-associated antigens may lead to better selectivity and efficacy in eliminating leukemic stem cells and blasts,representing a promising therapeutic strategy for AML. View Publication

SARS-CoV-2 Omicron variant has evolved into sublineages. Here,we compared the neutralization susceptibility and viral fitness of EG.5.1 and XBB.1.9.1. Serum neutralization antibody titer against EG.5.1 was 1.71-fold lower than that for XBB.1.9.1. However,there was no significant difference in virus replication between EG.5.1 and XBB.1.9.1 in human nasal organoids and TMPRSS2/ACE2 over-expressing A549 cells. No significant difference was observed in competitive fitness and cytokine/chemokine response between EG.5.1 and XBB.1.9.1. Both EG.5.1 and XBB.1.9.1 replicated more robustly in the nasal organoid from a younger adult than that from an older adult. Our findings suggest that enhanced immune escape contributes to the dominance of EG.5.1 over earlier sublineages. The combination of population serum susceptibility testing and viral fitness evaluation with nasal organoids may hold promise in risk assessment of upcoming variants. Utilization of serum specimens and nasal organoid derived from older adults provides a targeted risk assessment for this vulnerable population. Subject areas: Immunology,Immune response,Virology View Publication

N6‐methyladenosine (m6A) is an RNA modification involved in RNA processing and widely found in transcripts. In cancer cells,m6A is upregulated,contributing to their malignant transformation. In this study,we analyzed gene expression and m6A modification in cancer tissues,ducts,and acinar cells derived from pancreatic cancer patients using MeRIP‐seq. We found that dozens of RNAs highly modified by m6A were detected in cancer tissues compared with ducts and acinar cells. Among them,the m6A‐activated mRNA TCEAL8 was observed,for the first time,as a potential marker gene in pancreatic cancer. Spatially resolved transcriptomic analysis showed that TCEAL8 was highly expressed in specific cells,and activation of cancer‐related signaling pathways was observed relative to TCEAL8‐negative cells. Furthermore,among TCEAL8‐positive cells,the cells expressing the m6A‐modifying enzyme gene METTL3 showed co‐activation of Notch and mTOR signaling,also known to be involved in cancer metastasis. Overall,these results suggest that m6A‐activated TCEAL8 is a novel marker gene involved in the malignant transformation of pancreatic cancer. View Publication

BCR::ABL1-independent pathways contribute to primary resistance to tyrosine kinase inhibitor (TKI) treatment in chronic myeloid leukemia (CML) and play a role in leukemic stem cell persistence. Here,we perform ex vivo drug screening of CML CD34 + leukemic stem/progenitor cells using 100 single drugs and TKI-drug combinations and identify sensitivities to Wee1,MDM2,and BCL2 inhibitors. These agents effectively inhibit primitive CD34 + CD38 − CML cells and demonstrate potent synergies when combined with TKIs. Flow-cytometry-based drug screening identifies mepacrine to induce differentiation of CD34 + CD38 − cells. We employ genome-wide CRISPR-Cas9 screening for six drugs,and mediator complex,apoptosis,and erythroid-lineage-related genes are identified as key resistance hits for TKIs,whereas the Wee1 inhibitor AZD1775 and mepacrine exhibit distinct resistance profiles. KCTD5,a consistent TKI-resistance-conferring gene,is found to mediate TKI-induced BCR::ABL1 ubiquitination. In summary,we delineate potential mechanisms for primary TKI resistance and non-BCR::ABL1-targeting drugs,offering insights for optimizing CML treatment. View Publication

Pathogenic variants in MYH7 and MYBPC3 account for the majority of hypertrophic cardiomyopathy (HCM). Targeted drugs like myosin ATPase inhibitors have not been evaluated in children. We generate patient and variant-corrected iPSC-cardiomyocytes (CMs) from pediatric HCM patients harboring single variants in MYH7 ( V606M ; R453C ),MYBPC3 ( G148R ) or digenic variants ( MYBPC3 P955fs,TNNI3 A157V ). We also generate CMs harboring MYBPC3 mono- and biallelic variants using CRISPR editing of a healthy control. Compared with isogenic and healthy controls,variant-positive CMs show sarcomere disorganization,higher contractility,calcium transients,and ATPase activity. However,only MYH7 and biallelic MYBPC3 variant-positive CMs show stronger myosin-actin binding. Targeted myosin ATPase inhibitors show complete rescue of the phenotype in variant-positive CMs and in cardiac Biowires to mirror isogenic controls. The response is superior to verapamil or metoprolol. Myosin inhibitors can be effective in genotypically diverse HCM highlighting the need for myosin inhibitor drug trials in pediatric HCM. View Publication

Johnson,Ogishi,and Domingo-Vila et al. describe two siblings with inherited PD-L1 deficiency. Human PD-L1 deficiency underlies early-onset T1D,like PD-1 deficiency,but does not lead to fatal autoimmunity with extensive leukocytic dysregulation,unlike PD-1 deficiency. View Publication

MicroRNAs (miRNAs) are short non-coding RNAs that play crucial roles in gene regulation,exerting post-transcriptional silencing,thereby influencing cellular function,development,and disease. Traditional loss-of-function methods for studying miRNA functions,such as miRNA inhibitors and sponges,present limitations in terms of specificity,transient effects,and off-target effects. Similarly,CRISPR/Cas9-based editing of miRNAs using single guide RNAs (sgRNAs) also has limitations in terms of design space for generating effective gRNAs. In this study,we introduce a novel approach that utilizes CRISPR/Cas9 with dual guide RNAs (dgRNAs) for the rapid and efficient generation of short deletions within miRNA genomic regions. Through the expression of dgRNAs through single-copy lentiviral integration,this approach achieves over a 90% downregulation of targeted miRNAs within a week. We conducted a comprehensive analysis of various parameters influencing efficient deletion formation. In addition,we employed doxycycline (Dox)-inducible expression of Cas9 from the AAVS1 locus,enabling homogeneous,temporal,and stage-specific editing during cellular differentiation. Compared to miRNA inhibitory methods,the dgRNA-based approach offers higher specificity,allowing for the deletion of individual miRNAs with similar seed sequences,without affecting other miRNAs. Due to the increased design space,the dgRNA-based approach provides greater flexibility in gRNA design compared to the sgRNA-based approach. We successfully applied this approach in two human cell lines,demonstrating its applicability for studying the mechanisms of human erythropoiesis and pluripotent stem cell (iPSC) biology and differentiation. Efficient deletion of miR-451 and miR-144 resulted in blockage of erythroid differentiation,and the deletion of miR-23a and miR-27a significantly affected iPSC survival. We have validated the highly efficient deletion of genomic regions by editing protein-coding genes,resulting in a significant impact on protein expression. This protocol has the potential to be extended to delete multiple miRNAs within miRNA clusters,allowing for future investigations into the cooperative effects of the cluster members on cellular functions. The protocol utilizing dgRNAs for miRNA deletion can be employed to generate efficient pooled libraries for high-throughput comprehensive analysis of miRNAs involved in different biological processes. View Publication

Non-small cell lung cancer (NSCLC) is known for high relapse rates despite resection in early stages. Here,we present the results of a phase I clinical trial in which a dendritic cell (DC) vaccine targeting patient-individual neoantigens is evaluated in patients with resected NSCLC. Vaccine manufacturing is feasible in six of 10 enrolled patients. Toxicity is limited to grade 1–2 adverse events. Systemic T cell responses are observed in five out of six vaccinated patients,with T cell responses remaining detectable up to 19 months post vaccination. Single-cell analysis indicates that the responsive T cell population is polyclonal and exhibits the near-entire spectrum of T cell differentiation states,including a naive-like state,but excluding exhausted cell states. Three of six vaccinated patients experience disease recurrence during the follow-up period of 2 years. Collectively,these data support the feasibility,safety,and immunogenicity of this treatment in resected NSCLC. View Publication

Prenatal lethality associated with mouse knockout of Mettl16,a recently identified RNA N6-methyladenosine (m 6 A) methyltransferase,has hampered characterization of the essential role of METTL16-mediated RNA m 6 A modification in early embryonic development. Here,using cross-species single-cell RNA sequencing analysis,we found that during early embryonic development,METTL16 is more highly expressed in vertebrate hematopoietic stem and progenitor cells (HSPCs) than other methyltransferases. In Mettl16-deficient zebrafish,proliferation capacity of embryonic HSPCs is compromised due to G1/S cell cycle arrest,an effect whose rescue requires Mettl16 with intact methyltransferase activity. We further identify the cell-cycle transcription factor mybl2b as a directly regulated by Mettl16-mediated m 6 A modification. Mettl16 deficiency resulted in the destabilization of mybl2b mRNA,likely due to lost binding by the m 6 A reader Igf2bp1 in vivo. Moreover,we found that the METTL16-m 6 A- MYBL2 -IGF2BP1 axis controlling G1/S progression is conserved in humans. Collectively,our findings elucidate the critical function of METTL16-mediated m 6 A modification in HSPC cell cycle progression during early embryonic development. View Publication

Chemotherapy remains the mainstay treatment for triple-negative breast cancer (TNBC) due to the lack of specific targets. Given a modest response of immune checkpoint inhibitors in TNBC patients,improving immunotherapy is an urgent and crucial task in this field. CD73 has emerged as a novel immunotherapeutic target,given its elevated expression on tumor,stromal,and specific immune cells,and its established role in inhibiting anti-cancer immunity. CD73-generated adenosine suppresses immunity by attenuating tumor-infiltrating T- and NK-cell activation,while amplifying regulatory T cell activation. Chemotherapy often leads to increased CD73 expression and activity,further suppressing anti-tumor immunity. While debulking the tumor mass,chemotherapy also enriches heterogenous cancer stem cells (CSC),potentially leading to tumor relapse. Therefore,drugs targeting both CD73,and CSCs hold promise for enhancing chemotherapy efficacy,overcoming treatment resistance,and improving clinical outcomes. However,safe and effective inhibitors of CD73 have not been developed as of now. We used in silico docking to screen compounds that may be repurposed for inhibiting CD73. The efficacy of these compounds was investigated through flow cytometry,RT-qPCR,CD73 activity,cell viability,tumorsphere formation,and other in vitro functional assays. For assessment of clinical translatability,TNBC patient-derived xenograft organotypic cultures were utilized. We also employed the ovalbumin-expressing AT3 TNBC mouse model to evaluate tumor-specific lymphocyte responses. We identified quercetin and luteolin,currently used as over-the-counter supplements,to have high in silico complementarity with CD73. When quercetin and luteolin were combined with the chemotherapeutic paclitaxel in a triple-drug regimen,we found an effective downregulation in paclitaxel-enhanced CD73 and CSC-promoting pathways YAP and Wnt. We found that CD73 expression was required for the maintenance of CD44 high CD24 low CSCs,and co-targeting CD73,YAP,and Wnt effectively suppressed the growth of human TNBC cell lines and patient-derived xenograft organotypic cultures. Furthermore,triple-drug combination inhibited paclitaxel-enriched CSCs and simultaneously improved lymphocyte infiltration in syngeneic TNBC mouse tumors. Conclusively,our findings elucidate the significance of CSCs in impairing anti-tumor immunity. The high efficacy of our triple-drug regimen in clinically relevant platforms not only underscores the importance for further mechanistic investigations but also paves the way for potential development of new,safe,and cost-effective therapeutic strategies for TNBC. View Publication

Microglia,the brain’s resident immune cells,play vital roles in brain development,and disorders like Alzheimer’s disease (AD). Human iPSC-derived microglia (iMG) provide a promising model to study these processes. However,existing iMG generation protocols face challenges,such as prolonged differentiation time,lack of detailed characterization,and limited gene function investigation via CRISPR-Cas9. Our integrated toolkit for in-vitro microglia functional genomics optimizes iPSC differentiation into iMG through a streamlined two-step,20-day process,producing iMG with a normal karyotype. We confirmed the iMG’s authenticity and quality through single-cell RNA sequencing,chromatin accessibility profiles (ATAC-Seq),proteomics and functional tests. The toolkit also incorporates a drug-dependent CRISPR-ON/OFF system for temporally controlled gene expression. Further,we facilitate the use of multi-omic data by providing online searchable platform that compares new iMG profiles to human primary microglia: https://sherlab.shinyapps.io/IPSC-derived-Microglia/ . Our method generates iMG that closely align with human primary microglia in terms of transcriptomic,proteomic,and chromatin accessibility profiles. Functionally,these iMG exhibit Ca2 + transients,cytokine driven migration,immune responses to inflammatory signals,and active phagocytosis of CNS related substrates including synaptosomes,amyloid beta and myelin. Significantly,the toolkit facilitates repeated iMG harvesting,essential for large-scale experiments like CRISPR-Cas9 screens. The standalone ATAC-Seq profiles of our iMG closely resemble primary microglia,positioning them as ideal tools to study AD-associated single nucleotide variants (SNV) especially in the genome regulatory regions. Our advanced two-step protocol rapidly and efficiently produces authentic iMG. With features like the CRISPR-ON/OFF system and a comprehensive multi-omic data platform,our toolkit equips researchers for robust microglial functional genomic studies. By facilitating detailed SNV investigation and offering a sustainable cell harvest mechanism,the toolkit heralds significant progress in neurodegenerative disease drug research and therapeutic advancement. The online version contains supplementary material available at 10.1186/s13287-024-03700-9. View Publication