Persistent Expression Of Human Factor IX In Mice After Subcutaneous Transplantation Of Genetically Modified Human Umbilical Cord Mesenchymal Stem Cells

Background and Objectives:Hemophilia B is an X-linked bleeding disorder caused by a deficiency in functional coagulation factorⅨ.In the patients lacking FⅨ,the intrinsic coagulation pathway is disrupted,which may lead to lifelong spontaneous hemorrhages and prolonged bleeding after trauma or surgery.Current management,by intravenous infusion of clotting factor concentrates,either prophylactically or on demand,is a highly effective treatment,but clearly falls short of this long-term goal.The goal of gene therapy for genetic diseases is to bring about long-lasting expression of the missing or defective gene.Gene delivery system is necessary to be efficient,safe, non-immunogenic and allow for long-term gene expression.With the capacity of high proliferation and differentiation and low immunogenicity,human umbilical cord mesenchymal stem cells(HUCMSCs) are attractive target cells for ex vivo gene therapy of genetic diseases,including haemophilia B.In this study,we have develop a gene therapy project for haemophilia B which aims to express human factorⅨ(hFⅨ) in HUCMSCs.Methods:The retrovirus vectors that contain human factorⅨcDNA and part of intron 1 of hFⅨand the control ones were constructed and employed to prepare recombinant retroviruses.HUCMSCs infected with these viruses were selected with the neomycin analogue,G418 sulfate,and tested for expression of factorⅨby ELISA,western blot analysis.The biological activity was also determined by the clotting assay employing human factorⅨ-deficient plasma.Following xenografting of the transduced HUCMSCs into immunodeficient NOD/SCID mice,the hFⅨlevels in the serum were detected by ELISA. Results:Transduced cells produced biologically active hFⅨwith 100-130% activity in two-day culture supernatant and expressed hFⅨat levels of 2.68±0.36μg/10~6 cells/24h after G418 selection for 10 days.The secretion of hFⅨinto culture supernatant was confirmed by Western blot analysis.Genetically modified HUCMSCs(6×10~6 cells per mouse) were subcutaneously transplanted into NOD/SCID mice.At one week post-implantation,serum samples contained hFⅨat levels greater than 108.4 ng/ml.Circulating levels of hFⅨgradually decreased from 80.7ng/ml at 4 weeks post-implantation to 38.1ng/ml at 8 weeks.Conclusions:Our study demonstrates that genetically modified HUCMSCs can continuously secrete biologically active hFⅨfrom ectopic transplantation site in vivo,and can thus serve as an efficient drug delivery vehicle producing factorⅨin a somatic gene therapy for hemophilia B. Background and Objectives:Bmi-1 is a transcriptional repressor,which belongs to the Polycomb group family.The most widely known target of Bmi-1 is the INK4a locus,which encodes the p16 and p19ARF tumor suppressor proteins。Bmi-1 overexpression reduces INK4a expression BMI-1 has been shown to be indispensable for the self-renewal of neural and haematopoietic stem cells.It also has a crucial role in regulating the proliferative activity of stem and progenitor cells.It has been demonstrated that over-expression of Bmi-1 occurs in a variety of cancers,including several types of leukemia.Bmi-1 gene plays a key role in regulation of self-renewal by normal and leukemic stem cellsm.Leukemic cells lacking Bmi-1 underwent proliferation arrest and showed signs of differentiation and apoptosis.These findings led to the proposal of Bmi-1 as a potential target for therapeutic intervention in cancer.The purpose of this study is to investigate the effect of short hairpin RNA(shRNA) targeting Bmi-lon K562 cell line.Methods:The shRNA eukaryotic expression vector targeting Bmi-1 was constructed and transfected into K562 cells through lipofactamine 2000.The mRNA and protein level of Bmi-1 were detected by PCR and western blot.The proliferation of k562 after Bmi-1 silencing was measured using MTT assay and clone formation assay.The cell cycle was detected by flow cytometry.Results:Among the four shRNA,there was a shRNA which efficiently interfered with the expression of Bmi-1.The results of PCR and western blot validated that the Bmi-1 gene of K562 cells transfected with such a Bmi-1 shRNA was suppressed successfully.Although levels of Bmi-1 mRNA and protein were significantly reduced,delivery of this siRNAs had no effect on cell viability or growth.Flow cytometry analysis suggested that Bmi-1 inhibition did not affect the cell cycle.Conclusions:The suppression of Bmi-1 expression is not able to reduce proliferation of k562 cells,suggesting that there are some other parallel signalling pathways,which are fundamental for transformation and whose deregulation is independent of Bmi-1 over-expression.Bmi-1 over-expression may plays a secondary role in chronic myeloid leukemia transformation. Objectives:Idiopathic thrombocytopenic purpura(ITP) is a disease putatively related to abnormal immune function and auto-anti-platelet immunoglobulin.The polarization of Th1/Th2 toward Thl contributes to the pathogenesis of idiopathic thrombocytopenic purpura(ITP).Cytokines may play crucial roles in the pathogenesis of ITP.The purpose of this study is to investigate whether the interferon-gamma +874(A/T) and interleukin-4 (IL-4) variable number of tandern repeats(VNTR) in intron3 polymorphisms may be responsible in part for genetic susceptibility to ITP.Methods:Genotyping of IFNgamma +874A/T and IL-4 intron3 VNTR was performed in 196 patients with ITP and 128 healthy individuals by PCR-SSP and direct PCR respectively.Results:There was no association between IFNgamma+874A/T and IL-4 intron3 VNTR polymorphism and ITP risk when all patients as a group were analyzed.When the patients were subdivided into two groups:childhood ITP and adult ITP,no statistically differences were found in the genotype and allele frequencies of IFN-gamma +874A/T and IL-4 intron3 VNTR between the two groups and the controls.Similar results were observed between acute childhood ITP,chronic childhood ITP,acute adult ITP or chronic adult ITP and the controls.Conclusions:These polymorphisms were distributed similarly between the patients with ITP and the controls,demonstrating that these two candidate gene polymorphisms are not attributed to ITP susceptibility.