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Genetic diseases of blood cells are prime candidates for treatment through ex vivo gene editing of CD34+ hematopoietic stem/progenitor cells (HSPCs), and a variety of technologies have been proposed to treat these disorders. Sickle cell disease (SCD) is a recessive genetic disorder caused by a single-nucleotide polymorphism in the β-globin gene (HBB). Sickle hemoglobin damages erythrocytes, causing vasoocclusion, severe pain, progressive organ damage, and premature death. We optimize design and delivery parameters of a ribonucleoprotein (RNP) complex comprising Cas9 protein and unmodified single guide RNA, together with a single-stranded DNA oligonucleotide donor (ssODN), to enable efficient replacement of the SCD mutation in human HSPCs. Corrected HSPCs from SCD patients produced less sickle hemoglobin RNA and protein and correspondingly increased wild-type hemoglobin when differentiated into erythroblasts. When engrafted into immunocompromised mice, ex vivo treated human HSPCs maintain SCD gene edits throughout 16 weeks at a level likely to have clinical benefit. These results demonstrate that an accessible approach combining Cas9 RNP with an ssODN can mediate efficient HSPC genome editing, enables investigator-led exploration of gene editing reagents in primary hematopoietic stem cells, and suggests a path toward the development of new gene editing treatments for SCD and other hematopoietic diseases.

Objective Accumulating evidence from microbial studies have highlighted the modulatory roles of intestinal microbes in numerous human diseases, however, the shared microbial signatures across different diseases remain relatively unclear. Methods To consolidate existing knowledge across multiple studies, we performed meta-analyses of 17 disease types, covering 34 case–control datasets of 16S rRNA sequencing data, to identify shared alterations among different diseases. Furthermore, the impact of a microbial species, Lactobacillus salivarius , was established in a dextran sodium sulphate–induced colitis model and a collagen type II–induced arthritis mouse model. Results Microbial alterations among autoimmune diseases were substantially more consistent compared with that of other diseases (cancer, metabolic disease and nervous system disease), with microbial signatures exhibiting notable discriminative power for disease prediction. Autoimmune diseases were characterized by the enrichment of Enterococcus , Veillonella , Streptococcus and Lactobacillus and the depletion of Ruminococcus , Gemmiger , Oscillibacter , Faecalibacterium , Lachnospiracea incertae sedis , Anaerostipes , Coprococcus , Alistipes , Roseburia , Bilophila , Barnesiella , Dorea , Ruminococcus2 , Butyricicoccus , Phascolarctobacterium , Parabacteroides and Odoribacter , among others. Functional investigation of L. salivarius , whose genus was commonly enriched in numerous autoimmune diseases, demonstrated protective roles in two separate inflammatory mouse models. Conclusion Our study highlights a strong link between autoimmune diseases and the gut microbiota, with notably consistent microbial alterations compared with that of other diseases, indicating that therapeutic strategies that target the gut microbiome may be transferable across different autoimmune diseases. Functional validation of L. salivarius highlighted that bacterial genera associated with disease may not always be antagonistic, but may represent protective or adaptive responses to disease. [ABSTRACT FROM AUTHOR]

The equine herpesvirus type 8 (EHV-8) can cause significant economic losses in the global horses and donkey industry. The disease has been associated with abortion and respiratory symptoms. However, it is rare for a study to be reported about donkeys with neurological diseases induced by EHV-8 infection. In the present study, one 2-year-old male donkey, from a large-scale donkey farm in China, died with a severe neurological disorder. The causative agent, donkey/Shandong/10/2021 (GenBank accession: OL856098), was identified and isolated from the brain tissue of the dead donkey. Meanwhile, BALB/c mice were used as an animal model to evaluate the pathogenicity of the EHV-8 isolate. Our data showed that EHV-8 was positive in brains by PCR and immunohistochemistry, which induced typical viral encephalitis lesions in both donkey and mice consistent with clinical signs. For the first time, we reported that EHV-8 had been isolated from donkeys with a neurological illness in China, which is helpful to reveal the pathogenicity of EHV-8 in the donkey. [ABSTRACT FROM AUTHOR]

Background: The Zc3h8 gene encodes a protein with three zinc finger motifs in the C-terminal region. The protein has been identified as a component of the Little Elongation Complex, involved in transcription of small nuclear RNAs. ZC3H8 is overexpressed in a number of human and mouse breast cancer cell lines, and elevated mRNA levels are associated with a poorer prognosis for women with breast cancer.Methods: We used RNA silencing to decrease levels of expression in mouse mammary tumor cells and overexpression of ZC3H8 in cells derived from the normal mouse mammary gland. We measured characteristics of cell behavior in vitro, including proliferation, migration, invasion, growth in soft agar, and spheroid growth. We assessed the ability of these cells to form tumors in syngeneic BALB/c mice. ZC3H8 protein was visualized in cells using confocal microscopy.Results: Tumor cells with lower ZC3H8 expression exhibited decreased proliferation rates, slower migration, reduced ability to invade through a basement membrane, and decreased anchorage independent growth in vitro. Cells with lower ZC3H8 levels formed fewer and smaller tumors in animals. Overexpression of ZC3H8 in non-tumorigenic COMMA-D cells led to an opposite effect. ZC3H8 protein localized to both PML bodies and Cajal bodies within the nucleus. ZC3H8 has a casein kinase 2 (CK2) phosphorylation site near the N-terminus, and a CK2 inhibitor caused the numerous PML bodies and ZC3H8 to coalesce to a few larger bodies. Removal of the inhibitor restored PML bodies to their original state. A mutant ZC3H8 lacking the predicted CK2 phosphorylation site showed localization and numbers of ZC3H8/PML bodies similar to wild type. In contrast, a mutant constructed with a glutamic acid in place of the phosphorylatable threonine showed dramatically increased numbers of smaller nuclear foci.Conclusions: These experiments demonstrate that Zc3h8 expression contributes to aggressive tumor cell behavior in vitro and in vivo. Our studies show that ZC3H8 integrity is key to maintenance of PML bodies. The work provides a link between the Little Elongation Complex, PML bodies, and the cancer cell phenotype. [ABSTRACT FROM AUTHOR]