The Studies Of An Adenovirus Vector Retargeting Hematopoietic Cells And Induced Pluripotent Stem Cells

All cell components in blood are derived from the proliferation and differentiation of hematopoietic stem cells,which are also the most commonly used target cells for gene therapy.Recombinant adenovirus is the most widely used gene therapy vector in clinical trials,however,hematopoietic cells do not express adenovirus receptor(CAR),it is very inefficient to infect blood cells.i PSCs are the induced pluripotent stem cells that have the ability of self-renewal and pluripotent cells by reprogramming by transferring specific exogenous transcription factors into differentiated somatic cells,which also have broad development prospects and great significance in many scientific fields such as basic research and regenerative medicine.Peripheral blood cells are commonly used to induce i PSCs.We are committed to gene modification or modification of a variety of cells,including lymphocytes and i PSCs,by means of gene therapy,which will greatly enhance the application of gene therapy in clinical blood diseases.The aim of this study was to investigate the properties of recombinant adenovirus vectors and reprogrammed pluripotent stem cells induced by blood cells.Objective:(1)To study the efficient transfection of human adenovirus type 5 with fiber apical sphere reconstruction on lymphocytes.(2)To establish a system of hemocyte-induced pluripotent stem cells(i PSCs)and their differentiation into MSC.Methods:(1)first,the CMV promoter in pshuttle-cmv was replaced by human EF1 a promoter to generate shuttle plasmid p Sh5EF1 a.Secondly,the fibrous gene in the padeasy-1 skeleton plasmid was modified to generate a new skeleton plasmid of the hadv-5 vector.Subsequently,adenovirus plasmids were produced by homologous recombination using skeletal plasmids and linearized p Sh5 EG e.coli strain BJ5183.The adenovirus plasmids were digested by Pac I,recovered by ethanol precipitation,and transfected into 293 cells by Lipofectamine 3000.After three rounds of freezing and thawing,the saved virus was released and amplified in 293 cells.The amplified virus was purified by conventional Cs Cl overspeed centrifugation.Particle titer was determined by quantitative purification of viral genomic DNA and infection titer was determined by limited dilution assay of 293 cells.Four hematopoietic cell lines from U937,K562,Jurkat and hl-60,CD34+ from human umbilical cord blood,and T cells from human peripheral blood were selected to evaluate the gene transfer ability of four fiber-modified hadv-5 cells.The cells were infected and GFP fluorescence was analyzed2 days later.(2)5-10 ml fresh peripheral blood was diluted and gradient centrifuged,peripheral blood mononuclear cells were obtained for culture.Sendai virus vectors encoding OCT4,SOX2,KLF4,and c-myc were used to efficiently transfect a small number of lymphocytes and drive the fate of highly differentiated lymphocytes to the pluripotent stem cell state.3-4w continuously subculture the emerging i PSCs clones,and remove the heterozygous cells including differentiation,so as to obtain i PSCs that can maintain multi-competence in a long-term and stable manner.The morphological and iconic proteins by immunofluorescence localization were identified.The identified i PSCs were induced to differentiate into mesenchymal stem cells(MSC)using specific culture medium,and their cell morphology and iconic proteins were identified by flow cytometry.Results:(1)four first-generation adenovirus vectors were constructed,abbreviated as f35-eg,f11p-eg,hr-eg and cr-eg,respectively.All vectors transfected over 90% of K562 or Jurkat cells with a complex number of infection(MOI)of 500 virus particles per cell.All carriers except the hr-eg were able to transfect nearly 90% of CD34+ cells in cord blood or 80% of primary human T cells by 1000 MOI,and f11p-eg showed slight advantages over f35-eg and cr-eg.Adenovirus vector transmits CD4 + T cells more efficiently than CD8+ T cells.These vectors did not show cytotoxicity at MOI of up to1000vp/ cells because the infected and uninfected T cells maintained the same CD4 /CD8 ratio and cell growth rate.(2)MNC was successfully isolated and obtained,and sendai virus vector with four reprogramming factors was introduced into lymphocytes.Multiple clones with i PSCs morphology appeared within the expected time.After several subcultures,the iconic proteins(Nanog,oct-4,Sox2,SSEA4)were identified by immunofluorescence localization,and the results were all positive.Flow cytometry identification of multiple surface proteins of i PSCs-induced MSC showed that the expressions of CD11 b,CD19 and CD34 were all lower than 5%,and the expressions of CD73,CD90 and CD105 were all higher than 80%.Conclusion: HAd V-11 p fiber pseudotyped HAd V-5 could effectively transduce human T cells when human EF1 a promoter was used to control the expression of transgene,suggesting its possible application in T cell immunocellular therapy.The experimental system of i PSCs induced by blood cells and i PSCs differentiation into MSC was established,providing a frontier model for further research on gene therapy.