The Construction Of A Novel Vector Targeting To The HrDNA Locus With VEGF165 And Targeted Expression Of VEGF165 In Cell

Vascular endothelial growth factor (VEGF) is an angiogenicregulator to stimulate endothelial cell migration, proliferation andangiogenesis. VEGF gene therapy is a new promising approach to inducetherapeutic angiogenesis for the treatment of ischemic myocardial andlimb diseases. Recently, Clinical studies have demonstrated successfuloutcomes using plasmids, retroviruses, and adenoviruses.Selecting vector is one of the important factors in gene therapy.Indeed, among the universe of clinical gene transfer trials, the viral vectoris widely used, including retrovirus, adenovirus, or adenovirus associatedvirus. However, the safety of those vectors is poor and made it a seriousconcern. Besides immunogenicity caused by viral vectors, a furtherproblem with viral approaches is that viral vectors show a preference tointegrate into the transcription or control region of active genes, then itmay induce tumour and other disorders. Efficient and safe nonviralvectors have become of the subject many researchers pursued. Thus, theresearchers of National lab of medical genetics of China have constructeda new nonviral vector—hrDNA targeting vector: pHrneo, pHrneo is ahuman source gene vector that targets heterologous gene to the humanribosomal DNA (hrDNA)locus with an efficiency of integration up to10^-4-10^-5. In this study, pHrneo was chosen as the vector in the research of gene therapy with VEGF165. Human ribosomal RNA gene (rDNA)locates in D and G group chromosome. In human cells, there are about600-800 copies rDNA per cell. For the specific structure of rDNA, anexogenous gene inserting into ribosomal gene target site to destroy a copyof rDNA, there may be no effect on the cell physiological function.After amplified by PCR, the VEGF165 fragment, together withCMV, EGFP and BGHploy(A), was combined to construct a expressioncassette. Then the cassette was inserted into pHrneo vector to constructpHrneo-VEGF165. Through electroporation pHrneo-VEGF165 wastransferred into HT1080 cells. Finally we identified the homologousrecombinants by PCR, and tested the expression of VEGF165 in thehrDNA locus.The VEGF165 was successfully targeted into hrDNA locus ofHT-1080 by pHrneo-VEGF165, and the efficiency of site-specificintegration was 7.7×10^-5 The quantity of VEGF165 fusion protein was(1000±23) pg·10⁶ cells^-1·24 h^-1 tested by ELISA, the mRNA and fusionprotein of VEGF165 were detected by RT-PCR and Western-blottingrespectively. This research will provide new insights into the clinicalresearch of ischemic limb and myocardial diseases.