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

CRISPR-Cas9 genome editing often induces unintended,large genomic rearrangements,posing potential safety risks. However,there are no methods for mitigating these risks. Using long-read individual-molecule sequencing (IDMseq),we found the microhomology-mediated end joining (MMEJ) DNA repair pathway plays a predominant role in Cas9-induced large deletions (LDs). We targeted MMEJ-associated genes genetically and/or pharmacologically and analyzed Cas9-induced LDs at multiple gene loci using flow cytometry and long-read sequencing. Reducing POLQ levels or activity significantly decreases LDs,while depleting or overexpressing RPA increases or reduces LD frequency,respectively. Interestingly,small-molecule inhibition of POLQ and delivery of recombinant RPA proteins also dramatically promote homology-directed repair (HDR) at multiple disease-relevant gene loci in human pluripotent stem cells and hematopoietic progenitor cells. Our findings reveal the contrasting roles of RPA and POLQ in Cas9-induced LD and HDR,suggesting new strategies for safer and more precise genome editing. The online version contains supplementary material available at 10.1186/s12915-024-01896-z. View Publication

The use of genetic engineering to generate point mutations in induced pluripotent stem cells (iPSCs) is essential for studying a specific genetic effect in an isogenic background. We demonstrate that a combination of p53 inhibition and pro-survival small molecules achieves a homologous recombination rate higher than 90% using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in human iPSCs. Our protocol reduces the effort and time required to create isogenic lines. View Publication

Kidney organoids are an innovative tool in transplantation research. The aim of the present study was to investigate whether kidney organoids are susceptible for allo-immune attack and whether they can be used as a model to study allo-immunity in kidney transplantation. Human induced pluripotent stem cell-derived kidney organoids were co-cultured with human peripheral blood mononuclear cells (PBMC),which resulted in invasion of allogeneic T-cells around nephron structures and macrophages in the stromal cell compartment of the organoids. This process was associated with the induction of fibrosis. Subcutaneous implantation of kidney organoids in immune-deficient mice followed by adoptive transfer of human PBMC led to the invasion of diverse T-cell subsets. Single cell transcriptomic analysis revealed that stromal cells in the organoids upregulated expression of immune response genes upon immune cell invasion. Moreover,immune regulatory PD-L1 protein was elevated in epithelial cells while genes related to nephron differentiation and function were downregulated. This study characterized the interaction between immune cells and kidney organoids,which will advance the use of kidney organoids for transplantation research. View Publication

Early childhood tumours arise from transformed embryonic cells,which often carry large copy number alterations (CNA). However,it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains,which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNAs impair the specification of trunk neural crest (NC) cells and their sympathoadrenal derivatives,the putative cells-of-origin of NB. This effect is exacerbated upon overexpression of MYCN,whose amplification co-occurs with CNAs in NB. Moreover,CNAs potentiate the pro-tumourigenic effects of MYCN and mutant NC cells resemble NB cells in tumours. These changes correlate with a stepwise aberration of developmental transcription factor networks. Together,our results sketch a mechanistic framework for the CNA-driven initiation of embryonal tumours. Subject terms: Paediatric cancer,Stem cells,Disease model,Cancer genomics,Embryonal neoplasms View Publication

Hematopoiesis continues throughout life to produce all types of blood cells from hematopoietic stem cells (HSCs). Metabolic state is a known regulator of HSC self-renewal and differentiation,but whether and how metabolic sensor O -GlcNAcylation,which can be modulated via an inhibition of its cycling enzymes O -GlcNAcase (OGA) and O -GlcNAc transferase (OGT),contributes to hematopoiesis remains largely unknown. Herein,isogenic,single-cell clones of OGA -depleted (OGAi) and OGT -depleted (OGTi) human induced pluripotent stem cells (hiPSCs) were successfully generated from the master hiPSC line MUSIi012-A,which were reprogrammed from CD34 + hematopoietic stem/progenitor cells (HSPCs) containing epigenetic memory. The established OGAi and OGTi hiPSCs exhibiting an increase or decrease in cellular O -GlcNAcylation concomitant with their loss of OGA and OGT,respectively,appeared normal in phenotype and karyotype,and retained pluripotency,although they may favor differentiation toward certain germ lineages. Upon hematopoietic differentiation through mesoderm induction and endothelial-to-hematopoietic transition,we found that OGA inhibition accelerates hiPSC commitment toward HSPCs and that disruption of O -GlcNAc homeostasis affects their commitment toward erythroid lineage. The differentiated HSPCs from all groups were capable of giving rise to all hematopoietic progenitors,thus confirming their functional characteristics. Altogether,the established single-cell clones of OGTi and OGAi hiPSCs represent a valuable platform for further dissecting the roles of O -GlcNAcylation in blood cell development at various stages and lineages of blood cells. The incomplete knockout of OGA and OGT in these hiPSCs makes them susceptible to additional manipulation,i.e.,by small molecules,allowing the molecular dynamics studies of O -GlcNAcylation. View Publication

Hemangiosarcoma and angiosarcoma are soft-tissue sarcomas of blood vessel–forming cells in dogs and humans,respectively. These vasoformative sarcomas are aggressive and highly metastatic,with disorganized,irregular blood-filled vascular spaces. Our objective was to define molecular programs which support the niche that enables progression of canine hemangiosarcoma and human angiosarcoma. Dog-in-mouse hemangiosarcoma xenografts recapitulated the vasoformative and highly angiogenic morphology and molecular characteristics of primary tumors. Blood vessels in the tumors were complex and disorganized,and they were lined by both donor and host cells. In a series of xenografts,we observed that the transplanted hemangiosarcoma cells created exuberant myeloid hyperplasia and gave rise to lymphoproliferative tumors of mouse origin. Our functional analyses indicate that hemangiosarcoma cells generate a microenvironment that supports expansion and differentiation of hematopoietic progenitor populations. Furthermore,gene expression profiling data revealed hemangiosarcoma cells expressed a repertoire of hematopoietic cytokines capable of regulating the surrounding stromal cells. We conclude that canine hemangiosarcomas,and possibly human angiosarcomas,maintain molecular properties that provide hematopoietic support and facilitate stromal reactions,suggesting their potential involvement in promoting the growth of hematopoietic tumors. We demonstrate that hemangiosarcomas regulate molecular programs supporting hematopoietic expansion and differentiation,providing insights into their potential roles in creating a permissive stromal-immune environment for tumor progression. View Publication

Cohesin plays a crucial role in the organization of topologically-associated domains (TADs),which influence gene expression and DNA replication timing. Whether epigenetic regulators may affect TADs via cohesin to mediate DNA replication remains elusive. Here,we discover that the histone demethylase PHF2 associates with RAD21,a core subunit of cohesin,to regulate DNA replication in mouse neural stem cells (NSC). PHF2 loss impairs DNA replication due to the activation of dormant replication origins in NSC. Notably,the PHF2/RAD21 co-bound genomic regions are characterized by CTCF enrichment and epigenomic features that resemble efficient,active replication origins,and can act as boundaries to separate adjacent domains. Accordingly,PHF2 loss weakens TADs and chromatin loops at the co-bound loci due to reduced RAD21 occupancy. The observed topological and DNA replication defects in PHF2 KO NSC support a cohesin-dependent mechanism. Furthermore,we demonstrate that the PHF2/RAD21 complex exerts little effect on gene regulation,and that PHF2’s histone-demethylase activity is dispensable for normal DNA replication and proliferation of NSC. We propose that PHF2 may serve as a topological accessory to cohesin for cohesin localization to TADs and chromatin loops,where cohesin represses dormant replication origins directly or indirectly,to sustain DNA replication in NSC. View Publication

Adult T-cell leukemia/lymphoma (ATL) occurs after human T-cell leukemia virus type-1 (HTLV-1) infection with a long latency period exceeding several decades. This implies the presence of immune evasion mechanisms for HTLV-1-infected T cells. Although ATL cells have a CD4 + CD25 + phenotype similar to that of regulatory T cells (Tregs),they do not always possess the immunosuppressive functions of Tregs. Factors that impart effective immunosuppressive functions to HTLV-1-infected cells may exist. A previous study identified a new CD13 + Treg subpopulation with enhanced immunosuppressive activity. We,herein,describe the paired CD13 − (designated as MT-50.1) and CD13 + (MT-50.4) HTLV-1-infected T-cell lines with Treg-like phenotype,derived from the peripheral blood of a single patient with lymphoma-type ATL. The cell lines were found to be derived from HTLV-1-infected non-leukemic cells. MT-50.4 cells secreted higher levels of immunosuppressive cytokines,IL-10 and TGF-β,expressed higher levels of Foxp3,and showed stronger suppression of CD4 + CD25 − T cell proliferation than MT-50.1 cells. Furthermore,the CD13 inhibitor bestatin significantly attenuated MT-50.4 cell growth,while it did not for MT-50.1 cells. These findings suggest that CD13 expression may be involved in the increased Treg-like activity of MT-50.4 cells. Hence,MT-50.4 cells will be useful for in-depth studies of CD13 + Foxp3 + HTLV-1-infected cells. Subject terms: Cancer,Microbiology,Oncology View Publication

Chimeric antigen receptor (CAR) T cell therapy has emerged as a powerful therapeutic approach against a range of hematologic malignancies. While the incorporation of CD28 or 4-1BB costimulatory signaling domains into CARs revolutionized immune responses,there is an exciting prospect of further enhancing CAR functionality. Here,we investigated the design of CD19 CARs enriched with distinct Toll-like receptor 4 (TLR4),myeloid differentiation primary response 88 (MyD88),or Toll/IL-1 domain-containing adaptor-inducing interferon (IFN)-β (TRIF) costimulatory domains. Screening of various designs identified several candidates with no tonic activity but with increased CD19 target cell-dependent interleukin (IL)-2 production. Human T cells transduced with the selected CAR construct exhibited augmented hIL-2 and hIFN-γ induction and cytotoxicity when cocultured with CD19-positive lymphoma and solid-tumor cell lines. RNA sequencing (RNA-seq) analysis demonstrated the upregulation of some genes involved in the innate immune response and T cell activation and proliferation. In experiments on a xenogeneic solid-tumor mice model,MyD88 and TLR4 CAR T cells exhibited prolonged remission. This study demonstrates that the integration of a truncated TLR4 signaling costimulatory domain could provide immunotherapeutic potential against both hematologic malignancies and solid tumors. View Publication

Precise gene editing uncovers mis-splicing of BUBR1 and CDC27 in human SF3B1-mutant HSPCs,leading to activation of mitotic checkpoint and rendering the cells sensitive to CHK1 inhibitor prexasertib. View Publication

Hypoxia-activated prodrug (HAP) is a promising candidate for highly tumor-specific chemotherapy. However,the oxygenation heterogeneity and dense extracellular matrix (ECM) of tumor,as well as the potential resistance to chemotherapy,have severely impeded the resulting overall efficacy of HAP. A HAP potentiating strategy is proposed based on ultrasound responsive nanodroplets (PTP@PLGA),which is composed of protoporphyrin (PpIX),perfluoropropane (PFP) and a typical HAP,tirapazamine (TPZ). The intense vaporization of PFP upon ultrasound irradiation can magnify the sonomechanical effect,which loosens the ECM to promote the penetration of TPZ into the deep hypoxic region. Meanwhile,the PpIX enabled sonodynamic effect can further reduce the oxygen level,thus activating the TPZ in the relatively normoxic region as well. Surprisingly,abovementioned ultrasound effect also results in the downregulation of the stemness of cancer cells,which is highly associated with drug-refractoriness. This work manifests an ideal example of ultrasound-based nanotechnology for potentiating HAP and also reveals the potential acoustic effect of intervening cancer stem-like cells. The online version contains supplementary material available at 10.1186/s12951-024-02623-0. View Publication