Studies On Transferring Variant DHFR And GFP Fusion Gene Into Human Peripheral Blood Hematopoietic Cells By AAV

OBJECTIVE In gene therapy in which hematopoietic stem cell are regarded as target cell, how to transfer novel gene into static hematopoietic stem cells is still one of the most obstacles faced at present. The dihydrofolate reductase (DHFR) gene expression level is so low that it can抰 confrant the myelosuppression caused by anti-neoplastic drug methotrexate (MTX) in human hematopoietic stem cells. Taking adeno-associated virus (AAV) as vector, we design to transfer the human variant dihydrofolate reductase (L22Y/F3 1 R-mDHFR) gene and reporter green fluorescent proteins (GFP) gene into hematopoietic stem cells to protect human hematopoietic function, as well as to build a convenient, practical architecture to detect novel gene. METHODS Constructed rAAV/mDHFR-GFP vector and packaged it into recombinant viruses by 293 cells, and the recombiant viruses infected NIH3T3 cells and human peripheral blood CD34+ cells respectively. mDHFR-GFP cDNA were detected by PCR in NIH3T3 cells, as well as mDHFR-GFP mRNA were detected by RT-PCR in human peripheral blood CD34~ cells. An assay of MTT proved that mDHFR gene had expression in two kinds of cells with MTh culture, and there was some difference of 4 resistance to MTh between transferred gene group and untransfered group. The transferation rate were observed by flow cytometry and fluorescent microscope. RESULTS 1. Constructed recombinant AAV/mDHFR-GFP vector and it could express successfully. 2. Proved that AAV vector could transfer mDHFR-GFP cDNA into peripheral blood CD34+ cells by PCR. 3. RT-PCR proved that mDHFR and GFP mRNA existed in transferred peripheral blood CD34+ cells, therefor showed that transferred novel gene existed in the target cells precisely. 4. rAAV/mDHFR-GFP viruses infected NIH3T3 cells and human peripheral blood CD34+ cells respectively, the infection rate was about 20% and there were green fluorescence protein expression in peripheral blood stem and ancestor cells. 5. MTT proved that the resistance had been enhanced in a certain extent in transferred cells than untransffered cells. CONCLUSION AAV vector is able to transfer mDHFR-GFP fusion gene into peripheral blood CD34+ cells successfully, and transferred novel gene can express effectively in target cells, therefor produce functional protein which enhance the resistance to MTX of transferred cells. It needs to impove AAV package system and gene transferation technology to raise gene transferation rate.