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Background: LentiGlobin BB305 is a self‐inactivating lentiviral vector carrying a human β‐globin expressing cassette for treating β‐thalassemia. Initially, a 2 × 250 bp chicken Locus Control Region fragment of cHS4, functioning as an insulator, was placed at its ΔU3, which was removed after the first clinical trial led by a French team to avoid abnormal splicing, etc. This action could potentially lead to an increasing risk of the transcriptional read‐through rate driven by the β‐globin promoter to a significant level, posing a biosafety risk in clinical trials. Methods: In the present study, a read‐through reducing agent (C‐U+ or WPRE) was designed to be placed at the 3′ UTR of the β‐globin gene. The Enhancer Activities and/or Transcriptional Read‐Through (EATRT) rate at the mRNA level and the protein expression level regarding lentiviral preparation titer were examined. Results: We found that the insertion of the element (C‐U+ or WPRE) reduced the EATRT effectively by 53% or 41%, respectively. C‐U+ has less impact on virus package efficiency. Furthermore, there was no significant difference in the protein expression level after the C‐U+ or WPRE insertion. Conclusions: The results of the present study show that inserting C‐U+ or WPRE before the polyA sequence of the BB305 would reduce the EATRT rate at no cost of its expressing efficacy and viral preparation titers. Thus, we present an alternative improvement for a safer lentiviral vector for β‐thalassemia clinical trials. [ABSTRACT FROM AUTHOR]

Limb‐girdle muscular dystrophy recessive 1 (LGMDR1), previously known as LGMD2A, is a specific LGMD caused by a gene mutation encoding the calcium‐dependent neutral cysteine protease calpain‐3 (CAPN3). In our study, the compound heterozygosity with two missense variants c.635 T > C (p.Leu212Pro) and c.2120A > G (p.Asp707Gly) was identified in patients with LGMDR1. However, the pathogenicity of c.635 T > C has not been investigated. To evaluate the effects of this novel likely pathogenic variant to the motor system, the mouse model with c.635 T > C variant was prepared by CRISPR/Cas9 gene editing technique. The pathological results revealed that a limited number of inflammatory cells infiltrated the endomyocytes of certain c.635 T > C homozygous mice at 10 months of age. Compared with wild‐type mice, motor function was not significantly impaired in Capn3 c. 635 T > C homozygous mice. Western blot and immunofluorescence assays further indicated that the expression levels of the Capn3 protein in muscle tissues of homozygous mice were similar to those of wild‐type mice. However, the arrangement and ultrastructural alterations of the mitochondria in the muscular tissues of homozygous mice were confirmed by electron microscopy. Subsequently, muscle regeneration of LGMDR1 was simulated using cardiotoxin (CTX) to induce muscle necrosis and regeneration to trigger the injury modification process. The repair of the homozygous mice was significantly worse than that of the control mice at day 15 and day 21 following treatment, the c.635 T > C variant of Capn3 exhibited a significant effect on muscle regeneration of homozygous mice and induced mitochondrial damage. RNA‐sequencing results demonstrated that the expression levels of the mitochondrial‐related functional genes were significantly downregulated in the mutant mice. Taken together, the results of the present study strongly suggested that the LGMDR1 mouse model with a novel c.635 T > C variant in the Capn3 gene was significantly dysfunctional in muscle injury repair via impairment of the mitochondrial function. [ABSTRACT FROM AUTHOR]

Objectives: Mesoderm, derived from a new layer between epiblast and hypoblast during gastrulation, can differentiate into various tissues, including muscles, bones, kidneys, blood, and the urogenital system. However, systematic elucidation of mesoderm characteristics and specific markers remains a challenge. This study aims to screen and identify candidate genes important for mesoderm development. Materials and Methods: Cells originating from the three germ layers were obtained by laser capture microdissection, followed by microcellular RNA sequencing. Mesoderm‐specific differentially expressed genes (DEGs) were identified by using a combination of three bioinformatics pipelines. Candidate mesoderm‐specific genes expression were verified by real‐time quantitative polymerase chain reaction analysis and immunohistochemistry. Functional analyses were verified by ESCs‐EBs differentiation and colony‐forming units (CFUs) assay. Results: A total of 1962 differentially expressed mesoderm genes were found, out of which 50 were candidate mesoderm‐specific DEGs which mainly participate in somite development, formation of the primary germ layer, segmentation, mesoderm development, and pattern specification process by GO analysis. Representative genes Cdh2, Cdh11, Jag1, T, Fn‐1, and Pcdh7 were specifically expressed in mesoderm among the three germ layers. Pcdh7 as membrane‐associated gene has hematopoietic‐relevant functions identified by ESCs‐EBs differentiation and CFUs assay. Conclusions: Spatial transcriptomic profiling with multi‐method analysis and confirmation revealed candidate mesoderm progenitors. This approach appears to be efficient and reliable and can be extended to screen and validate candidate genes in various cellular systems. [ABSTRACT FROM AUTHOR]

Objectives: Early embryo development is dependent on the regulation of maternal messages stored in the oocytes during the maternal‐to‐zygote transition. Previous studies reported variability of oocyte competence among different inbred mouse strains. The present study aimed to identify the maternal transcripts responsible for early embryonic development by comparing transcriptomes from oocytes of high‐ or low‐ competence mouse strains. Materials and Methods: In vitro fertilization embryos from oocytes of different mouse strains were subject to analysis using microarrays, RNA sequencing, real‐time quantitative PCR (RT‐qPCR) analysis, Western blotting, and immunofluorescence. One candidate gene, Prkce, was analysed using Prkce knockout mice, followed by a cRNA rescue experiment. Results: The fertilization and 2‐cell rate were significantly higher for FVB/NJ (85.1% and 82.0%) and DBA/2J (79.6% and 76.7%) inbred mouse strains than those for the MRL/lpr (39.9% and 35.8%) and 129S3 (35.9% and 36.6%) strains. Thirty‐nine differentially expressed genes (DEGs) were noted, of which nine were further verified by RT‐qPCR. Prkce knockout mice showed a reduced 2‐cell rate (Prkce+/+ 80.1% vs. Prkce−/− 32.4%) that could be rescued by Prkce cRNA injection (2‐cell rate reached 76.7%). Global transcriptional analysis revealed 143 DEGs in the knockout mice, which were largely composed of genes functioning in cell cycle regulation. Conclusions: The transcription level of maternal messages such as Prkce in mature oocytes is associated with different 2‐cell rates in select inbred mouse strains. Prkce transcript levels could serve as a potential biomarker to characterize high‐quality mature oocytes. [ABSTRACT FROM AUTHOR]

Mental retardation is the main clinical manifestation of Down syndrome (DS), and neural abnormalities occur during the early embryonic period and continue throughout life. Tc1, a model mouse for DS, carries the majority part of the human chromosome 21 and has multiple neuropathy phenotypes similar to patients with DS. To explore the mechanism of early neural abnormalities of Tc1 mouse, induced pluripotent stem (iPS) cells from Tc1 mice were obtained, and genome‐wide gene expression and methylation analysis were performed for Tc1 and wild‐type iPS cells. Our results showed hypermethylation profiles for Tc1 iPS cells, and the abnormal genes were shown to be related to neurodevelopment and distributed on multiple chromosomes. In addition, important genes involved in neurogenesis and neurodevelopment were shown to be downregulated in Tc1 iPS cells. In short, our study indicated that genome‐wide hypermethylation leads to the disordered expression of genes associated with neurodevelopment in Tc1 mice during early development. Overall, our work provided a useful reference for the study of the molecular mechanism of nervous system abnormalities in DS. [ABSTRACT FROM AUTHOR]

Nanog is a well-known transcription factor that plays a fundamental role in stem cell self-renewal and the maintenance of their pluripotent cell identity. There remains a large data gap with respect to the spectrum of the key pluripotency transcription factors' interaction partners. Limited information is available concerning Nanog-associated RNA-binding proteins (RBPs), and the intrinsic protein-RNA interactions characteristic of the regulatory activities of Nanog. Herein, we used an improved affinity protocol to purify Nanog-interacting RBPs from mouse embryonic stem cells (ESCs), and 49 RBPs of Nanog were identified. Among them, the interaction of YBX1 and ILF3 with Nanog mRNA was further confirmed by in vitro assays, such as Western blot, RNA immunoprecipitation (RIP), and ex vivo methods, such as immunofluorescence staining and fluorescent in situ hybridization (FISH), MS2 in vivo biotin-tagged RNA affinity purification (MS2-BioTRAP). Interestingly, RNAi studies revealed that YBX1 and ILF3 positively affected the expression of Nanog and other pluripotency-related genes. Particularly, downregulation of YBX1 or ILF3 resulted in high expression of mesoderm markers. Thus, a reduction in the expression of YBX1 and ILF3 controls the expression of pluripotency-related genes in ESCs, suggesting their roles in further regulation of the pluripotent state of ESCs. [ABSTRACT FROM AUTHOR]

Human amniotic fluid derived progenitor cells (hAFPCs) may be multipotent and can be considered a potential tool in the field of cell therapy for haemophilia B. Their capacity to express human coagulation factor IX (hFIX) after transduction and their fate after in utero transplantation is unknown. hAFPCs isolated from second trimester pregnancies were assessed for their phenotypic markers, multilineage capacity, and expression of hFIX after transduction. Their engraftment potential was analysed in a mouse model after in utero transplantation at embryonic day 12.5. Immunohistochemistry, fluorescence in situ, ELISA and PCR were used to assess post-transplant chimeras. hAFPCs expressed several pluripotent markers, including NANOG, SOX2, SSEA4 and TRA-1-60, and could differentiate into adipocytes and osteocytes. In vitro, after transduction with hFIX and EGFP cDNAs, constitutive hFIX protein expression and clotting activity were found. Engraftment was achieved in various foetal tissues after in utero transplantation. Safe engraftment without oncogenesis was confirmed, with low donor cell levels, but persistent engraftment, into different organs (liver, heart and lung) through to 12 weeks of age. Transgenic expression of circulating hFIX was detected in recipient mice for up to 12 weeks. hAFPCs can be engrafted long-term in immunocompetent mice after in utero transplantation. Thus, cell transplantation approaches using genetically engineered hAFPCs may prove valuable for the prenatal treatment for haemophilia B. [ABSTRACT FROM AUTHOR]

Background Major improvements have been made progressively on human immunodeficiency virus (HIV)-1 based lentiviral vectors to minimize the probability of replication-competent lentivirus formation. This includes the deletion of U3 promoter and the use of packaging cells, which has increased their potential for use in gene therapy and other in vivo applications. However, the risk of forming replication-competent lentiviruses remains. Methods We investigated the use of Cre- loxP mediation with the insertion of the transgene-expressing cassette in ΔU3 to remove additional parts of the HIV-1 backbone upon cre expression, after integration. This, leads to deletion of the packaging signal, primer binding site and Rev response element, including cre itself. Results This approach left a split truncated form of long terminal repeat flanked by a loxP and a transgene-expressing cassette in the genome, which made replication-competent lentivirus formation almost impossible. This self-deletion vector could stably express transgenes both in cell lines and transgenic mice with only modest losses of viral titer. The maximum size of the inserts was approximately 3 kb, which was sufficient for most transgenic applications. Moreover, the addition of some enhancer blocking agents downstream of the transgene could reduce the probability of transcriptional read-through in transfected 293T cells. Conclusions Our approach could improve the biosafety of lentiviral vectors, thus improving their potential application for use in clinical trials and other in vivo applications. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Background: Lentiviral vectors (LVs) have enhancer activity and/or transcriptional read‐through (EATRT) properties that can lead to the activation of adjacent genes. Consequently, patients may be at increased risk for adverse effects if such vectors are used clinically. Methods: In the present study, we assessed the abilities of different "pro‐LV"‐like constructs with respect to decreasing its EATRT, including the "pro‐LV" vector bearing a chimeric ΔLTR of the human foamy virus R‐U5 region replaced by that of an LV (HF). Results: By analyzing the EATRT of "pro‐LV" constructs transfected in 293T cells, we observed that the inclusion of the first 400 bp of the chicken β‐globin locus HS4 insulator core sequence oriented in the reverse direction (C–) combined with two copies of the simian virus 40 upstream‐sequence element (U) at the ΔU3 of ΔLTR region of "pro‐LV" tended to shield the adjacent genomic sequences, such that the EATRT rate was lower than when either of the C– or U was included in the "pro‐LV". Moreover, upon transduction, the pro‐HF appears to reduce the EATRT rate in the chromosomes of 293T (by 80%) and human peripheral blood mononuclear cells (PBMCs) (by 75%) compared to when pro‐LV C–U was included (with a 60% and 89% reduction in 293T and PBMCs, respectively). The HF construct had a significant reduction of viral biological titer compared tiowhen the pro‐LV C–U was used in 293T cells. Conclusions: The results of the present study provide an important basis for the clinical applicability of LVs in gene and cell therapy. [ABSTRACT FROM AUTHOR]

Objectives: This study explored whether TALENs‐mediated non‐homologous end joining (NHEJ) targeting the mutation site can correct the aberrant β‐globin RNA splicing, and ameliorate the β‐thalassaemia phenotype in β654 mice. Material and methods: TALENs vectors targeted to the human β‐globin gene (HBB) IVS2‐654C >T mutation in a mouse model were constructed and selected to generate double heterozygous TALENs+/β654 mice. The gene editing and off‐target effects were analysed by sequencing analysis. β‐globin expression was identified by RT‐PCR and Western blot analysis. Various clinical indices including haematologic parameters and tissue pathology were examined to determine the therapeutic effect in these TALENs+/β654 mice. Results: Sequencing analysis revealed that the HBBIVS2‐654C >T point mutation was deleted in over 50% of the TALENs+/β654 mice tested, and off‐target effects were not detected. RT‐PCR and Western blot analysis confirmed the expression of normal β‐globin in TALENs+/β654 mice. The haematologic parameters were significantly improved as compared with their affected littermates. The proportion of nucleated cells in bone marrow was considerably decreased, splenomegaly with extramedullary haematopoiesis was reduced, and significant decreases in iron deposition were seen in spleen and liver of the TALENs+/β654 mice. Conclusion: These results suggest effective treatment of the anaemia phenotype in TALENs+/β654 mice following deletion of the mutation site by TALENs, demonstrating a simple and straightforward strategy for gene therapy of β654‐thalassaemia in the future. [ABSTRACT FROM AUTHOR]