Highly efficient Runx1 enhancer eR1-mediated genetic engineering for fetal, child and adult hematopoietic stem cells

A cis-regulatory genetic element which targets gene expression to stem cells, termed stem cell enhancer, serves as a molecular handle for stem cell-specific genetic engineering. Here we show the generation and characterization of a tamoxifen-inducible CreERT2 transgenic (Tg) mouse employing previously identified hematopoietic stem cell (HSC) enhancer for Runx1, eR1 (+24m). Kinetic analysis of labeled cells after tamoxifen injection and transplantation assays revealed that eR1-driven CreERT2 activity marks dormant adult HSCs which slowly but steadily contribute to unperturbed hematopoiesis. Fetal and child HSCs which are uniformly or intermediately active were also efficiently targeted. Notably, a gene ablation at distinct developmental stages, enabled by this system, resulted in different phenotypes. Similarly, an oncogenic Kras induction at distinct ages caused different spectrums of malignant diseases. These results demonstrate that the eR1-CreERT2 Tg mouse serves as a powerful resource for the analyses of both normal and malignant HSCs at all developmental stages.

Naturally Selected Anti-CD7 CAR-T Cells without Additional Genetic Manipulations As a Potentially Superior Therapy for T-Cell Malignancies

Introduction To prevent CAR-T fratricide, anti-CD7 CAR (7CAR) T cells used for treating T-cell malignancies are often modified by CD7 ablation via CRISPR/CAS9 gene editing or by co-expression of a CD7-specific protein expression blocker. Both methods require additional genetic manipulations of CAR-T. Here we transduce 7CAR into bulk T cells without CD7 disruption and thereafter allow CAR-T cells to emerge in vitro after fratricidal "natural selection". The biological characteristics of these naturally selected anti-CD7 CAR (NS7CAR) T cells and their potential advantages in treating patients with T-cell malignancies are described. Methods The percentage of CD3 +CD7 - T cells in peripheral blood from either healthy donors (HDs) or patients (PTs) were determined by flow cytometry. Peripheral bulk T cells were positively selected using CD3 magnetic beads, and peripheral CD7 - T cells were negatively selected using CD7 magnetic beads. To avoid contamination from malignant T cells, patients only with CD3 -CD7 + T cell blasts were included in this study. The 7CAR gene cassette comprising of the cDNA of a CD7-specific antibody sequence fused to the coding sequences for the CD8TM-41BB-CD3z signal domains, and the T2A-linked tEGFR was cloned into a lentiviral vector backbone under the control of an EF1α promoter. 7CAR lentiviral transduction of bulk T cells (NS7CAR) or CD7 - T cells (Neg7CAR) were performed two days after CD3/CD28 dynabeads activation. CD7-ablated 7CAR T cells (KO7CAR) were derived by electroporation of bulk T cells with CD7-targeting Cas9-gRNA RNP 24 hours before 7CAR transduction. CAR-T cells were routinely kept in culture for 12 days. The levels of CD7 mRNA, protein, and surface expression were determined respectively by qualitative/quantitative reverse transcription PCR, Western blotting, and flow cytometry. Iv vitro cytotoxic activity for CD7 + tumor cell lines was tested using a flow-cytometry-based cytotoxicity assay. NSG mice engrafted with CCRF-CEM-luciferase cells were used as an animal model to validate the activities of CD7 CAR. Results Three approaches for generating anti-CD7 CAR-T cells were compared: NS7CAR (fratricidal natural selection from bulk T cells after 7CAR transduction), Neg7CAR (7CAR transduction of purified CD7-negative T cells) and KO7CAR (Cas9 RNP CD7 gene ablation). While CD7 - T cells were detectable in HDs of all ages (9.48±0.96%, n=13), we observed a significant increase of this cell population in T-cell acute lymphoblastic leukemia PTs (15.98±0.57%, n=13) (Fig 1A). We next tested the feasibility of using bulk T cells to generate naturally occurring 7CAR T cells without CD7 gene ablation or protein blockage. Three days after 7CAR lentiviral transduction, purified bulk peripheral T cells had a rapid and dramatic phenotypic transition from CD7 + CAR - to CD7 -CAR +(Fig 1B). Although fratricide led to a much lower expansion and viability of 7CAR T cells compared to T- cells without 7CAR transduction, approximately 80% of the 7CAR T cells were viable, making further studies feasible. After 12 days of culture, 7CAR T cells from PTs displayed stronger expansion potential (Fig 1C) and contained a larger CD8 + subpopulation (Fig 1D) as compared to cells derived from HDs. In comparison to Neg7CAR and KO7CAR, the final NS7CAR product displayed a lower expansion capability, but contained a higher percentage of CAR + cells, a larger CD8 +subset and an increased central memory phenotype (Table 2). Interestingly, although the final cells from all three products had no surface CD7 expression, mRNA and total protein were only detected from NS7CAR, but not from Neg7CAR or KO7CAR. Additionally, NS7CAR showed superior cytotoxicity and cytokine release in an in vitro functional test (Fig 1E). In the animal model, the NS7CAR conferred robust protection against leukemia progression with marked reduction in leukemia cell burden in the first two weeks after CAR T- cells injection (Fig 1F&G). Conclusion Among the three approaches, the NS7CAR T cells was significantly enriched in CAR + cells and contained a higher percentage of CD8 + central memory T cells. Importantly, our data indicate that autologous PBMCs from patients were superior to PBMCs of healthy donors in yielding sufficient NS7CAR T cells for therapeutic needs. An investigator-initiated trial is currently ongoing to test the feasibility, efficacy, and safety of NS7CAR T cells for treating T-cell acute lymphoblastic leukemia. Figure 1 Figure 1. Disclosures Liu: SenlangBio: Current Employment. Ba: SenlangBio: Current Employment. Li: SenlangBio: Current holder of individual stocks in a privately-held company.

Delayed implantation induced by letrozole in mice

Implantation timing is key for a successful pregnancy. Short delay in embryo implantation caused by targeted gene ablation produced a cascading problem in the later stages of the pregnancy. Although several delayed implantation models have been established in wild mice, almost none of them is suitable for investigating the delay on the late events of pregnancy. Here, we report a new delayed implantation model established by the intraperitoneally administration of letrozole at 5 mg/kg body weight on the day 3 of pregnancy. In these mice, initiation of implantation was induced at will by the injection of estradiol (E2). When the estradiol (3 ng) was injected on day 4 of pregnancy (i.e., without delay), the embryo implantation restarted, and the pregnancy continued normally. However, high dose of estrogen (25 ng) caused compromised implantation. We also found that only 67% of the female mice could be pregnant normally and finally gave birth when the injection of estradiol (3 ng) was on day 5 of pregnancy (i.e., one day delay). Most of the failed pregnancies had impaired decidualization, decreased plasma progesterone levels and compromised angiogenesis. Progesterone supplementation could rescue decidualization failure in the mice. Collectively, we established a new model of delayed implantation by letrozole, which can be easily used to study the effect and mechanisms of delay of embryo implantation on the progression of late pregnancy events.

GDF15 induces immunosuppression via CD48 on regulatory T cells in hepatocellular carcinoma

BackgroundA better understanding of the molecular mechanisms that manifest in the immunosuppressive tumor microenvironment (TME) is crucial for developing more efficacious immunotherapies for hepatocellular carcinoma (HCC), which has a poor response to current immunotherapies. Regulatory T (Treg) cells are key mediators of HCC-associated immunosuppression. We investigated the selective mechanism exploited by HCC that lead to Treg cells expansion and to find more efficacious immunotherapies.MethodsWe used matched tumor tissues and blood samples from 150 patients with HCC to identify key factors of Treg cells expansion. We used mass cytometry (CyTOF) and orthotopic cancer mouse models to analyze overall immunological changes after growth differentiation factor 15 (GDF15) gene ablation in HCC. We used flow cytometry, coimmunoprecipitation, RNA sequencing, mass spectrum, chromatin immunoprecipitation and Gdf15–/–, OT-I and GFP transgenic mice to demonstrate the effects of GDF15 on Treg cells and related molecular mechanism. We used hybridoma technology to generate monoclonal antibody to block GDF15 and evaluate its effects on HCC-associated immunosuppression.ResultsGDF15 is positively associated with the elevation of Treg cell frequencies in patients wih HCC. Gene ablation of GDF15 in HCC can convert an immunosuppressive TME to an inflammatory state. GDF15 promotes the generation of peripherally derived inducible Treg (iTreg) cells and enhances the suppressive function of natural Treg (nTreg) cells by interacting with a previously unrecognized receptor CD48 on T cells and thus downregulates STUB1, an E3 ligase that mediates forkhead box P3 (FOXP3) protein degradation. GDF15 neutralizing antibody effectively eradicates HCC and augments the antitumor immunity in mouse.ConclusionsOur results reveal the generation and function enhancement of Treg cells induced by GDF15 is a new mechanism for HCC-related immunosuppression. CD48 is the first discovered receptor of GDF15 in the immune system which provide the possibility to solve the molecular mechanism of the immunomodulatory function of GDF15. The therapeutic GDF15 blockade achieves HCC clearance without obvious adverse events.

Bromodomain-containing protein BRPF1 is a therapeutic target for liver cancer

Epigenetic deregulation plays an essential role in hepatocellular carcinoma (HCC) progression. Bromodomains are epigenetic “readers” of histone acetylation. Recently, bromodomain inhibitors have exhibited promising therapeutic potential for cancer treatment. Using transcriptome sequencing, we identified BRPF1 (bromodomain and PHD finger containing 1) as the most significantly upregulated gene among the 43 bromodomain-containing genes in human HCC. BRPF1 upregulation was significantly associated with poor patient survival. Gene ablation or pharmacological inactivation of BRPF1 significantly attenuated HCC cell growth in vitro and in vivo. BRPF1 was involved in cell cycle progression, senescence and cancer stemness. Transcriptome sequencing revealed that BRPF1 is a master regulator controlling the expression of multiple key oncogenes, including E2F2 and EZH2. We demonstrated that BRPF1 activated E2F2 and EZH2 expression by facilitating promoter H3K14 acetylation through MOZ/MORF complex. In conclusion, BRPF1 is frequently upregulated in human HCCs. Targeting BRPF1 may be an approach for HCC treatment.

The myotendinous junction marker collagen XXII enables zebrafish postural control learning and optimal swimming performance through its force transmission activity

Although the myotendinous junction (MTJ) is essential for skeletal muscle integrity, its contribution to skeletal muscle function remains largely unknown. Here, we show that CRISPR-Cas9-mediated gene ablation of the MTJ marker col22a1 in zebrafish identifies two distinctive phenotypic classes: class 1 individuals reach adulthood with no overt muscle phenotype while class 2 display severe movement impairment and eventually dye before metamorphosis. Yet mutants that are unequally affected are all found to display defective force transmission attributed to a loss of ultrastructural integrity of the MTJ and myosepta, though with distinct degrees of severity. The behavior-related consequences of the resulting muscle weakness similarly reveal variable phenotypic expressivity. Movement impairment at the critical stage of swimming postural learning eventually causes class 2 larval death by compromising food intake while intensive exercise is required to uncover a decline in muscle performance in class 1 adults. By confronting MTJ gene expression compensation and structural, functional and behavioral insights of MTJ dysfunction, our work unravels variable expressivity of col22a1 mutant phenotype. This study also underscores COL22A1 as a candidate gene for myopathies associated with dysfunctional force transmission and anticipates a phenotypically heterogeneous disease.

Early life trauma leads to violent behavior and its inheritance by impairing local thyroid hormone availability in brain

Violent behavior is an aberrant form of aggression that has detrimental impact on health and society. Early life trauma triggers adulthood violence and criminality, though molecular mechanisms remain elusive. Here, we provide brain region specific transcriptome profiles of peripubertal stress (PPS) exposed adult violent male and resilient female mice. We identify transthyretin (TTR) as a key regulator of PPS induced violent behavior and its intergenerational inheritance. TTR mediated long-term perturbation in hypothalamic thyroid hormone (TH) availability contributed to male violent behavior without affecting circulating hormone. Ttr gene ablation in hypothalamus impaired local TH signaling including levels of TH transporters (Mct8, Oatp1c1), deiodinase 2 (DIO2) and TH responsive genes (Nrgn, Trh and Hr). Violent behavior and impaired TTR-TH signaling was also inherited in F1 male progenies. Further, we deciphered Ttr promoter hyper methylation in hypothalamus of violent males across generations. Our findings reveal that trauma during puberty trigger lasting violent behavior by epigenetic programming of TTR and consequent impaired local thyroid availability in brain. TTR-TH signaling in hypothalamus can serve as potential target in reversal of violent behavior.

Kcnj16 (Kir5.1) Gene Ablation Causes Subfertility and Increases the Prevalence of Morphologically Abnormal Spermatozoa

The ability of spermatozoa to swim towards an oocyte and fertilize it depends on precise K+ permeability changes. Kir5.1 is an inwardly-rectifying potassium (Kir) channel with high sensitivity to intracellular H+ (pHi) and extracellular K+ concentration [K+]o, and hence provides a link between pHi and [K+]o changes and membrane potential. The intrinsic pHi sensitivity of Kir5.1 suggests a possible role for this channel in the pHi-dependent processes that take place during fertilization. However, despite the localization of Kir5.1 in murine spermatozoa, and its increased expression with age and sexual maturity, the role of the channel in sperm morphology, maturity, motility, and fertility is unknown. Here, we confirmed the presence of Kir5.1 in spermatozoa and showed strong expression of Kir4.1 channels in smooth muscle and epithelial cells lining the epididymal ducts. In contrast, Kir4.2 expression was not detected in testes. To examine the possible role of Kir5.1 in sperm physiology, we bred mice with a deletion of the Kcnj16 (Kir5.1) gene and observed that 20% of Kir5.1 knock-out male mice were infertile. Furthermore, 50% of knock-out mice older than 3 months were unable to breed. By contrast, 100% of wild-type (WT) mice were fertile. The genetic inactivation of Kcnj16 also resulted in smaller testes and a greater percentage of sperm with folded flagellum compared to WT littermates. Nevertheless, the abnormal sperm from mutant animals displayed increased progressive motility. Thus, ablation of the Kcnj16 gene identifies Kir5.1 channel as an important element contributing to testis development, sperm flagellar morphology, motility, and fertility. These findings are potentially relevant to the understanding of the complex pHi- and [K+]o-dependent interplay between different sperm ion channels, and provide insight into their role in fertilization and infertility.

Emerging Next-Generation Target for Cancer Immunotherapy Research: The Orphan Nuclear Receptor NR2F6

Additional therapeutic targets suitable for boosting anti-tumor effector responses have been found inside effector CD4+ and CD8+ T cells. It is likely that future treatment options will combine surface receptor and intracellular protein targets. Utilizing germline gene ablation as well as CRISPR/Cas9-mediated acute gene mutagenesis, the nuclear receptor NR2F6 (nuclear receptor subfamily 2 group F member 6, also called Ear-2) has been firmly characterized as such an intracellular immune checkpoint in effector T cells. Targeting this receptor appears to be a strategy for improving anti-tumor immunotherapy responses, especially in combination with CTLA-4 and PD-1. Current preclinical experimental knowledge firmly validates the immune checkpoint function of NR2F6 in murine tumor models, which provides a promising perspective for immunotherapy regimens in humans in the near future. While the clinical focus remains on the B7/CD28 family members, protein candidate targets such as NR2F6 are now being investigated in laboratories around the world and in R&D companies. Such an alternative therapeutic approach, if demonstrated to be successful, could supplement the existing therapeutic models and significantly increase response rates of cancer patients and/or expand the reach of immune therapy regimens to include a wider range of cancer entities. In this perspective review, the role of NR2F6 as an emerging and druggable target in immuno-oncology research will be discussed, with special emphasis on the unique potential of NR2F6 and its critical and non-redundant role in both immune and tumor cells.