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Fetuses with homozygous [alpha]-thalassemia usually die at the third trimester of pregnancy or soon after birth. Hence, the disease could potentially be a target for fetal gene therapy. We have previously established a mouse model of [alpha]-thalassemia. These mice mimic the human [alpha]-thalassemic conditions and can be used as preclinical models for fetal gene therapy. We tested a lentiviral vector containing the HS 2, 3, and 4 of the [beta]-LCR, a central polypurine tract element, and the [beta]-globin gene promoter directing either the EGFP or the human [alpha]-globin gene. We showed that the GFP expression was erythroid-specific and detected in BFU-E colonies and the erythroid progenies of CFU-GEMM. For in utero gene delivery, we did yolk sac vessel injection at midgestation of mouse embryos. The recipient mice were analyzed after birth for human [alpha]-globin gene expression. In the newborn, human [alpha]-globin gene expression was detected in the liver, spleen, and peripheral blood. The human [alpha]-globin gene expression was at the peak at 3-4 months, when it reached 20% in some recipients. However, the expression declined at 7 months. Colony-forming assays in these mice showed low abundance of the transduced human [alpha]-globin gene in their BFU-E and CFU-GEMM and the lack of its transcript. Thus, lentiviral vectors can be an effective vehicle for delivering the human [alpha]-globin gene into erythroid cells in utero, but, in the mouse model, delivery at late midgestation could not transduce hematopoietic stem cells adequately to sustain gene expression. in utero gene transfer | lentiviral vector | yolk sac vessel injection

Periodontitis is an inflammatory disease resulting from subgingival microorganisms. Human periodontal ligament stem cells (hPDLSCs) can be applied in periodontal tissue regeneration. This study investigated the effect of hPDLSC-derived extracellular vesicles (EVs) on periodontitis. hPDLSC-derived EVs were isolated and identified. The murine model of periodontitis was established by ligation, and the cell model of periodontitis was established by treatment of macrophages with lipopolysaccharide (LPS). The effects of EVs on macrophage pyroptosis and periodontal inflammatory injury were measured by the means of HE staining, detection of LDH content, CCK-8 assay, Calcein-AM/PI staining, ELISA, Western blot, as well as measurement of caspase-1, SOD, and MDA. miR-590-3p expression was detected using RT-qPCR. miR-590-3p expression was then intervened to validate the effect of miR-590-3p on macrophage pyroptosis. The binding relationship between miR-590-3p and TLR4 was verified using dual-luciferase assay. Functional rescue experiment was performed to validate the role of TLR4 in macrophage pyroptosis. The results showed that inflammatory levels and macrophage pyroptosis were enhanced in the in vivo and in vitro models of periodontitis, evidenced by the increased NLRP3, GSDMD-N, caspase-1, IL-1β, IL-18, TNF-α, and MDA and decreased IL-10 and SOD. EVs alleviated periodontal inflammatory injury and macrophage pyroptosis. Physiologically, EVs carried miR-590-3p into macrophages to upregulate miR-590-3p expression and thereby suppress TLR4 transcription. miR-590-3p silencing or TLR4 overexpression reduced the inhibitory effect of EVs on macrophage pyroptosis. Collectively, EVs carried miR-590-3p into macrophages to subsequently inhibit TLR4 transcription, thereby reducing macrophage pyroptosis and alleviating periodontal inflammatory injury. [ABSTRACT FROM AUTHOR]