Preliminary Study Of Correction Of Murine Hemophilia B By Hydrodynamic Gene Dlivery And Site-specific Genomic Integration

Preliminary study of Correction of Murine Hemophilia B by Hydrodynamic Gene Dlivery and Site-specific Genomic IntegrationBackground and Objective:Hemophilia B is a sex-linked hemorrhagic disease resulting from deficiency in coagulation factor IX. The main clinical manifestation of the disease is unpredictable, recurrent, spontaneous bleeding in soft tissues and/or major joints. Recurrent bleeding in large joints usually leads to crippling arthropathies in a majority of severely affected patients. The clinical severity of hemophilia B corresponds to the level of circulating FIX. Current replacement therapy has significantly reduced the frequency of bleeding episodes and subsequent joint disease, and markedly improved the life expectancy of patients with hemophilia. However, the high cost of purified factor products makes life long prophylactic infusion impractical, and plasma-derived FIX would enable the patients to be at high risk for infection from contaminant viral pathogens. Generally, the FIX gene can be delivered via either non-viral or viral mediation. Recently, researchers pay more and more attention to non-viral vector. The site-specific integrase from phiC31 bacteriophage functions in mammaliancells and is being applied for genetic engineering, including gene therapy. In mammalian cells, the enzyme mediates integration of plasmids bearing attB into pseudo attP sites in host genome. Aneja and coworkers found that foreign plasmid expression in lung could be persistent when it co-injected with the phiC31-encoding plasmid. The purpose of our current study is to determine whether the plasmid bearing attB and hFIX coding sequence could insert into mice genome and persistently express hFIX when co-injected with the integrase.Methods:Firstly, we constructed the plasmid attB-hFIX-pIRES2-EGFP which bears attB site and hFIX coding sequence. It was proved that this plasmid could express hFIX through in vitro experiments. Then plasmid attB-hFIX-pIRES2-EGFP and CMV-int expressing integrase was co-injected by rapid infusion of a large-volume solution into the tail vein of hemophilia B mice. The mice injected attB-hFTX-pIRES2-EGFP alone served as controls. ELISA was performed to determine hFIX serum levels of hemophilia B mice. Correction of coagulation in vivo after plasmids injection was assessed by bleeding time assay. Genomic integration of foreign plasmid was determined by nested PCR.Results:The plasmid attB-hFIX-pIRES2-EGFP was successfully constructed and could express hFIX in vitro. The hemophilia B mice produced 1533±239 ng/ml hFIX 1 day after injection of the hFIX encoding plasmid and human FIX significantly corrected the bleeding diathesis of hemophilia B mice as measured by in vivo clotting assays. But, no matter co-injected with CMV-Int or not, the hFIX levels decreased to background level in 10 days after injection. Nested-PCR results indicated that the integrase phiC31 resulted in the integration of the plasmid in mouse liver chromosomes.Conclusion:Integrase phiC31 can catalyze recombination between 34bp attB and pseudo-attP. Human FIX driven by CMV promoter can be transiently and highly expressed afer hydrodynamic injection, but rapidly silenced no matter the insertion into genome or not. So, it is necessary to optimize the plasmid used in gene therapy according the target organ. Construction and Identification of a Novel Adeno-Integrase Hybrid System for Hemophilia BBackground and Objective:There are two kinds of vectors for hemophilia gene therapy:viral vector and non-viral vector. Viral vectors usually display high transduction efficiency of the target cell in vitro and in vivo, but generate humoral immune response and insertional mutagenesis. In contrast, non-viral vector systems are less immunogenic and easy to prepare, but delivery efficiency of foreign DNA is low. So we try to construct an adenovirus hybrid system with high transduction efficiencies and site-specific integration.Methods:By a serious of DNA manipulation, we constructed the hybrid system, including two adenovirus vectors. One vector contains loxp flanked transgene expression cassette, in which there are hFIX and DsRed coding sequences and attB for phiC31 recognization. The other vector carries Cre and phiC31 gene. We also constructed vectors only expressing Cre or phiC31 as controls.293A cells were transfected with the adenoviral vectors by Lipofectamine 2000 and the expression of target genes was identified by fluorescence microscope and RT-PCR.Results:After being identified by PCR, restriction enzyme digestion and sequencing, the adeno-integrase hybrid system was successfully constructed. The system can express RFP, GFP, hFIX, Cre and phiC31 in 293A cells in vitro.Conclusion:The adeno-integrase hybrid system was successfully constructed, which lay a good foundation for further investigation of its treatment effect. Thl (CXCL10) and Th2 (CCL2) Chemokine Expression in Patients with Primary Immune Thrombocytopenia and their Clinical ImplicationsBackground:Immune thrombocytopenia (ITP) is an organ-specific autoimmune disorder characterized by accelerated platelet destruction. The pathophysiology of ITP remains unclear. The imbalance of Thl/Th2 polarization might play important role in the pathogenesis of ITP. Th1 chemokine CXCL10 and Th2 chemokine CCL2 have been studied in several autoimmune diseases, but the status of these chemokines in ITP is still unknown.Objective:To determine the expression of CXCL10 and CCL2, and their receptors, CXCR3, CCR4 and CCR2 in ITP patients, and make a preliminary study of the pathogenic roles of these factors in ITP.Methods:Plasma samples from 49 patients with ITP, and 24 normal healthy subjects were assayed for CXCL10 and CCL2 plasma concentration by enzyme linked immunosorbent assay. Real-time quantitative PCR was performed to determine the mRNA expression of these chemokine and their receptors in the PBMNC of 24 normal controls and 28 active ITP patients as well as splenocytes of 9 ITP patients. The expression of CCR4 and CXCR3 in CD4+T cells from 10 ITP patients and 10 normal controls was compared by Real-time quantitative PCR.Results:The CXCL10 levels in the plasma samples from patients with active ITP were significantly higher than those from healthy controls (p=0.007), and decreased to normal levels in remission ITP. In contrast, CCL2 levels were similar in active patients, remission and control subjects. PBMNC of active patients expressed more CXCLIO mRNA (p=0.031) but less CCR2 mRNA (p=0.005). Lower peripheral platelet count correlated with higher CXCL10 levels and CXCL10/CCL2 ratios.Conclusion:Our study demonstrates that plasma levels of CXCL10 and CXC10/CCL2 ratio are higher in patients with active ITP than in healthy donors, and that CXCL10 might be a pathogenic factor of this disorder.