Xiuli Gong

A novel transgenic mouse model produced from lentiviral germline integration for the study of β-thalassemia gene therapy

Lentiviral-mediated gene therapy has been successfully applied in the treatment of β-thalassemia. In this study transgenic mice with stable expression of a lentivirus carrying the human β-globin gene were obtained. These animals provide a useful model to investigate the stable effect of gene therapy in β-thalassemia. Background β-thalassemia is one of the most common genetic diseases in the world and requires extensive therapy. Lentiviral-mediated gene therapy has been successfully exploited in the treatment of β-thalassemia and showed promise in clinical application. Using a human β-globin transgenic mouse line in a β-thalassemia diseased model generated with a lentiviral-mediated approach, we investigate the stable therapeutic effect on a common thalassemia syndrome. Design and Methods Human β-globin gene lentiviral vector was constr ucted, followed by subzonal microinjection into single-cell embryos of βIVS-2-654-thalassemia mice to generate a transgenic line. Human β-globin gene expression was examined with RT-PCR, Western-blotting and ELISA. The hematologic parameters and tissue pathology were investigated over time in founder mice and their off-spring. Results Transgenic mice with stable expression of the lentivirus carrying human β-globin gene were obtained. A marked improvement in red blood cell indices and a dramatic reduction in red blood cell anisocytosis, poikilocytosis and target cells were observed. Nucleated cell proportion was greatly decreased in bone marrow, and splenomegaly with extramedullary hematopoiesis was ameliorated. Iron deposition in liver was also reduced. There was a two-fold increase in the survival rate of the βIVS-2-654 mice carrying human β-globin transgene. Significantly, the germline integration of the lentiviral construct was obtained and stable hematologic phenotype correction was observed over the next two generations of the transgenic mice. Conclusions The generation of human β-globin transgenic mice in a βIVS-2-654-thalassemia mouse mediated with lentiviral vectors provides a useful model and offers an attractive means to investigate the transgenic stable therapeutic effect in β-thalassemia.

Induction of protection against foot-and-mouth disease virus in cell culture and transgenic suckling mice by miRNA targeting integrin αv receptor.

Foot-and-mouth disease virus (FMDV) is an RNA virus that causes a highly contagious disease in domestic and wild cloven-hoofed animals. Although vaccination has been used to protect animals against FMDV, there are shortcomings in the efficacy of the available vaccines. RNA interference (RNAi) is triggered by small RNA molecules, including short interfering RNAs and microRNAs (miRNAs), and the use of RNAi-based methods have demonstrated promise as an alternative method of controlling the transmission of FMDV. However, the method of delivery, short duration of siRNA and miRNA in vivo, and the genetic variability of FMDV confound the use of RNAi-based strategies for FMDV control. FMDV has been shown to exploit host-cell integrins as cell-surface receptors to initiate infection. We selected the gene for the integrin αv subunit as an RNAi target, and constructed three αv-specific miRNA expression plasmids. The effects of these miRNAs on FMDV infection were examined in PK-15 cells and transgenic suckling mice. In PK-15 cells, the expression of the αv-specific miRNAs significantly inhibited the expression of integrin αv receptor and decreased FMDV infection. The transgenic mice were generated by integrating the αv-specific miRNA expression cassette using pronuclear microinjection. When challenged with a dose of FMDV ten times greater than the LD50, the survival rate of transgenic suckling mice was approximately six-fold higher than that of their non-transgenic littermates, indicating that the interference of the miRNAs significantly reduced FMDV infection in the transgenic mice. This is the first report of limiting FMDV attachment to cellular receptors using miRNA-mediated gene knock down of cell-surface receptors to significantly reduce FMDV infection in cell culture and transgenic suckling mice.

The Mesenchymal Stem Cells Derived from Transgenic Mice Carrying Human Coagulation Factor VIII Can Correct Phenotype in Hemophilia A Mice

Hemophilia A (HA) is an inherited X-linked recessive bleeding disorder caused by coagulant factor VIII (FVIII) deficiency. Previous studies showed that introduction of mesenchymal stem cells (MSCs) modified by FVIII-expressing retrovirus may result in phenotypic correction of HA animals. This study aimed at the investigation of an alternative gene therapy strategy that may lead to sustained FVIII transgene expression in HA mice. B-domain-deleted human FVIII (hFVIIIBD) vector was microinjected into single-cell embryos of wild-type mice to generate a transgenic mouse line, from which hFVIIIBD-MSCs were isolated, followed by transplantation into HA mice. RT-PCR and real-time PCR analysis demonstrated the expression of hFVIIIBD in multi-organs of recipient HA mice. Immunohistochemistry showed the presence of hFVIIIBD positive staining in multi-organs of recipient HA mice. ELISA indicated that plasma hFVIIIBD level in recipient mice reached its peak (77 ng/mL) at the 3rd week after implantation, and achieved sustained expression during the 5-week observation period. Plasma FVIII activities of recipient HA mice increased from 0% to 32% after hFVIIIBD-MSCs transplantation. APTT (activated partial thromboplastin time) value decreased in hFVIIIBD-MSCs transplanted HA mice compared with untreated HA mice (45.5 s vs. 91.3 s). Our study demonstrated an effective phenotypic correction in HA mice using genetically modified MSCs from hFVIIIBD transgenic mice.

Hematopoietic stem cell engraftment by early-stage in utero transplantation in a mouse model

A novel intrauterine transplantation (IUT) approach was developed to improve the efficiency of engraftment of hematopoietic stem cells (HSCs). HSCs with a green fluorescent protein (GFP) reporter gene were transplanted in utero on days 12.5, 13.5 and 14.5 post coitum (p.c.). The degree of chimerism of donor cells in recipient newborn mice was examined using fluorescent microscopy, polymerase chain reaction (PCR), fluorescence-activated cell sorting (FACS), and fluorescence in situ hybridization (FISH) analyses. Microscopic examination revealed the presence of green fluorescent signal in the peripheral blood of the chimeric mice. The highest survival rate (47%) as well as the highest chimerism rate (73%) were achieved by our new approach in the newborn mice that were subjected to in utero transplantation (IUT) on day 12.5 p.c. (E12.5) compared to the conventional IUT method. FACS analysis indicated that 1.55+/-1.10% of peripheral blood cells from the newborn mice were GFP-positive donor cells. FISH showed that cells containing the donor-specific GFP sequence were present in the bone marrow (BM) of the chimeric mice. Thus, the efficiency of chimera production with this new method of IUT was significantly improved over the existing IUT techniques and instruments.

A self-deletion lentiviral vector to reduce the risk of replication-competent virus formation

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.

Association of differential and site-dependent CpG methylation and diverse expression of DNA methyltransferases with the tissue-specific expression of human β-globin gene in transgenic mice

Expression of human locus control region (LCR) and β-globin promoter has been recognized as an important factor in time- and tissue-specific expression event. DNA methylation can affect the transcriptional activity of specific genes. To investigate the methylation mechanism in the regulation of LCR and promote expression, this study used a transgenic mouse strain generated previously, in which the hematopoietic-specific expression of the EGFP was driven by human β-globin promoter and under the control of LCR, to examine the CpG methylation pattern in various tissues. The results showed the inverse correlation between the methylated extent and the levels of gene expression in all tested tissues. We also found that the methylated extent of the 10 examined CpG sites was biased along their positions and is more efficient near the transcription start site. Real-time quantitative RT-PCR analysis of DNA methyltransferases (DNMTs) transcripts showed that Dnmt3a and Dnmt3b expressed with a very low level in the hematopoietic tissues that was coincident with the relative higher EGFP expression in these tissues, indicating that the differential expression of DNMTs contributed to the tissue-specific methylated patterns which caused the diverse gene expression in various tissues. These findings provide significant clues to elucidate the mechanism of the regulation on tissue-specific expression of genes.

Engraftment of genetically modified human amniotic fluid-derived progenitor cells to produce coagulation factor IX after in utero transplantation in mice.

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.

A Herpes Simplex Virus Thymidine Kinase-Induced Mouse Model of Hepatocellular Carcinoma Associated with Up-Regulated Immune-Inflammatory-Related Signals

Inflammation and fibrosis in human liver are often precursors to hepatocellular carcinoma (HCC), yet none of them is easily modeled in animals. We previously generated transgenic mice with hepatocyte-specific expressed herpes simplex virus thymidine kinase (HSV-tk). These mice would develop hepatitis with the administration of ganciclovir (GCV). However, our HSV-tk transgenic mice developed hepatitis and HCC tumor as early as six months of age even without GCV administration. We analyzed the transcriptome of the HSV-tk HCC tumor and hepatitis tissue using microarray analysis to investigate the possible causes of HCC. Gene Ontology (GO) enrichment analysis showed that the up-regulated genes in the HCC tissue mainly include the immune-inflammatory and cell cycle genes. The down-regulated genes in HCC tumors are mainly concentrated in the regions related to lipid metabolism. Gene set enrichment analysis (GSEA) showed that immune-inflammatory-related signals in the HSV-tk mice are up-regulated compared to those in Notch mice. Our study suggests that the immune system and inflammation play an important role in HCC development in HSV-tk mice. Specifically, increased expression of immune-inflammatory-related genes is characteristic of HSV-tk mice and that inflammation-induced cell cycle activation maybe a precursory step to cancer. The HSV-tk mouse provides a suitable model for the study of the relationship between immune-inflammation and HCC, and their underlying mechanism for the development of therapeutic application in the future.

Treatment of β654-thalassaemia by TALENs in a mouse model

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.