Epigenetic inactivation of ERF reactivates γ-globin expression in ß-thalassemia.

The fetal-to-adult hemoglobin switch is regulated in a developmental stage-specific manner and reactivation of fetal hemoglobin (HbF) has therapeutic implications for treatment of ß-thalassemia and sickle cell anemia, two major global health problems. Although significant progress has been made in our understanding of the molecular mechanism of the fetal-to-adult hemoglobin switch, the mechanism of epigenetic regulation of HbF silencing remains to be fully defined. Here, we performed whole-genome bisulfite sequencing and RNA sequencing analysis of the bone marrow-derived GYPA+ erythroid cells from ß-thalassemia-affected individuals with widely varying levels of HbF groups (HbF ≥ 95th percentile or HbF ≤ 5th percentile) to screen epigenetic modulators of HbF and phenotypic diversity of ß-thalassemia. We identified an ETS2 repressor factor encoded by ERF, whose promoter hypermethylation and mRNA downregulation are associated with high HbF levels in ß-thalassemia. We further observed that hypermethylation of the ERF promoter mediated by enrichment of DNMT3A leads to demethylation of γ-globin genes and attenuation of binding of ERF on the HBG promoter and eventually re-activation of HbF in ß-thalassemia. We demonstrated that ERF depletion markedly increased HbF production in human CD34+ erythroid progenitor cells, HUDEP-2 cell lines, and transplanted NCG-Kit-V831M mice. ERF represses γ-globin expression by directly binding to two consensus motifs regulating γ-globin gene expression. Importantly, ERF depletion did not affect maturation of erythroid cells. Identification of alterations in DNA methylation of ERF as a modulator of HbF synthesis opens up therapeutic targets for ß-hemoglobinopathies.

The mouse resource at National Resource Center for Mutant Mice.

Mouse models are essential for dissecting disease mechanisms and defining potential drug targets. There are more than 18,500 mouse strains available for research communities in National Resource Center for Mutant Mice (NRCMM) of China, affiliated with Model Animal Research Center of Nanjing University and Gempharmatech Company. In 2019, Gempharmatech launched the Knockout All Project (KOAP) aiming to generate null mutants and gene floxed strains for all protein-coding genes in mouse genome within 5 years. So far, KOAP has generated 8,004 floxed strains and 9,769 KO (knockout) strains (updated to Oct, 2021). NRCMM also created hundreds of Cre transgenic lines, mutant knock-in models, immuno-deficient models, and humanized mouse models. As a member of the international mouse phenotyping consortium (IMPC), NRCMM provides comprehensive phenotyping services for mouse models. In summary, NRCMM will continue to support biomedical community with new mouse models as well as related services.

Fast and efficient generation of a full-length balancer chromosome by a single Cre/loxP recombination event.

Balancer chromosomes, primarily discovered and used in Drosophila melanogaster, are valuable tools to maintain lethal mutations in a particular genomic segment. Full-length balancer chromosomes would be particularly useful because of the capacity to maintain whole genomic traits. However, murine full-length balancer chromosomes generated via a single Cre/loxP recombination are still not demonstrated. In this study, we developed a novel mouse strain with full-length inverted chromosome 17 (Ch17Inv balancer) via a single Cre/loxP recombination event in mES cells. The Ch17Inv balancer mice are viable and phenotypically normal. When bred with other strains, the haplotype of chromosome 17 can be stably maintained as determined by the high throughput SNPs assay. Interestingly, we found that the recombination events were efficiently reduced within the inverted region but not eliminated. The method established in this study can be applied to generate other full-length balancer chromosomes. Moreover, the Ch17Inv balancer strain would be a valuable resource to maintain the entire chromosome 17 from different donor strains.

Technical considerations and strategies for generating and optimizing humanized mouse tumor models in immuno-oncology research.

The field of cancer immunotherapy has experienced significant progress, resulting in the emergence of numerous biological drug candidates requiring in vivo efficacy testing and a better understanding of their mechanism of action (MOA). Humanized immune system (HIS) models are valuable tools in this regard. However, there is a lack of systematic guidance on HIS modeling. To address this issue, the present study aimed to establish and optimize a variety of HIS models for immune-oncology (IO) study, including genetically engineered mouse models and HIS models with human immune components reconstituted in severely immunocompromised mice. The efficacy and utility of these models were tested with several marketed or investigational IO drugs according to their MOA, followed by immunophenotypic analysis and efficacy evaluation. The results of the present study demonstrated that the HIS models responded to various IO drugs as expected and that each model had unique niches, utilities and limitations. Researchers should carefully choose the appropriate models based on the MOA and the targeted immune cell populations of the investigational drug. The present study provides valuable methodologies and actionable technical guidance on designing, generating or utilizing appropriate HIS models to address specific questions in translational IO.

HX009, a novel BsAb dual targeting PD1 x CD47, demonstrates potent anti-lymphoma activity in preclinical models.

Both PD1/PD-L1 and CD47 blockades have demonstrated limited activity in most subtypes of NHL save NK/T-cell lymphoma. The hemotoxicity with anti-CD47 agents in the clinic has been speculated to account for their limitations. Herein we describe a first-in-class and rationally designed bispecific antibody (BsAb), HX009, targeting PD1 and CD47 but with weakened CD47 binding, which selectively hones the BsAb for tumor microenvironment through PD1 interaction, potentially reducing toxicity. In vitro characterization confirmed: (1) Both receptor binding/ligand blockade, with lowered CD47 affinity; (2) functional PD1/CD47 blockades by reporter assays; (3) T-cell activation in Staphylococcal-enterotoxin-B-pretreated PBMC and mixed-lymphocyte-reaction. In vivo modeling demonstrated antitumor activity in Raji-B and Karpass-229-T xenograft lymphomas. In the humanized mouse syngeneic A20 B-lymphoma (huCD47-A20) HuGEMM model, which has quadruple knocked-in hPD1xhPD-L1xhCD47xhSIRPα genes and an intact autologous immune-system, a contribution of effect is demonstrated for each targeted biologic (HX008 targeting PD1 and SIRPα-Fc targeting CD47), which is clearly augmented by the dual targeting with HX009. Lastly, the expression of the immune-checkpoints PD-L1/L2 and CD47 seemed co-regulated among a panel of lymphoma-derived-xenografts, where HX009 maybe more effective in those with upregulated CD47. Our data warrants HX009's further clinical development for treating NHLs.

Characterization and functional analysis of atl, a novel gene encoding autolysin in Streptococcus suis.

Streptococcus suis serotype 2 (S. suis 2) is an important swine and human pathogen responsible for septicemia and meningitis. A novel gene, designated atl and encoding a major autolysin of S. suis 2 virulent strain HA9801, was identified and characterized in this study. The Atl protein contains 1,025 amino acids with a predicted molecular mass of 113 kDa and has a conserved N-acetylmuramoyl-l-alanine amidase domain. Recombinant Atl was expressed in Escherichia coli, and its bacteriolytic and fibronectin-binding activities were confirmed by zymography and Western affinity blotting. Two bacteriolytic bands were shown in the sodium dodecyl sulfate extracts of HA9801, while both were absent from the atl inactivated mutant. Cell chains of the mutant strain became longer than that of the parental strain. In the autolysis assay, HA9801 decreased to 20% of the initial optical density (OD) value, while the mutant strain had almost no autolytic activity. The biofilm capacity of the atl mutant was reduced ∼30% compared to the parental strain. In the zebrafish infection model, the 50% lethal dose of the mutant strain was increased up to 5-fold. Furthermore, the adherence to HEp-2 cells of the atl mutant was 50% less than that of the parental strain. Based on the functional analysis of the recombinant Atl and observed effects of atl inactivation on HA9801, we conclude that Atl is a major autolysin of HA9801. It takes part in cell autolysis, separation of daughter cells, biofilm formation, fibronectin-binding activity, cell adhesion, and pathogenesis of HA9801.

Identification of a Chromosome 1 Substitution Line B6-Chr1BLD as a Novel Hyperlipidemia Model via Phenotyping Screening.

Hyperlipidemia is a chronic disease that seriously affects human health. Due to the fact that traditional animal models cannot fully mimic hyperlipidemia in humans, new animal models are urgently needed for basic drug research on hyperlipidemia. Previous studies have demonstrated that the genomic diversity of the wild mice chromosome 1 substitution lines was significantly different from that of laboratory mice, suggesting that it might be accompanied by phenotypic diversity. We first screened the blood lipid-related phenotype of chromosome 1 substitution lines. We found that the male HFD-fed B6-Chr1BLD mice showed more severe hyperlipidemia-related phenotypes in body weight, lipid metabolism and liver lesions. By RNA sequencing and whole-genome sequencing results of B6-Chr1BLD, we found that several differentially expressed single nucleotide polymorphism enriched genes were associated with lipid metabolism-related pathways. Lipid metabolism-related genes, mainly including Aida, Soat1, Scly and Ildr2, might play an initial and upstream role in the abnormal metabolic phenotype of male B6-Chr1BLD mice. Taken together, male B6-Chr1BLD mice could serve as a novel, polygenic interaction-based hyperlipidemia model. This study could provide a novel animal model for accurate clinical diagnosis and precise medicine of hyperlipidemia.