Gene-modified MSCS By RAAV2-eGFP Could Evade The Specific Immune Response Against Wide-type AAV2and Effectively Transduce The Target Gene When Allotransplanted Into The Rat’s Brain

Background:Promoting angiogenesis is one of the major treapeutic strategies for ischemic stroke. Many foctors (such as VEGF or Ang-1) have been applied for gene therapy reasearch, and the Adeno-Associated Virus(AAV) Vectors are the most commonly used vectors. As several clinical trials carried out, it was found that one of the major challenges facing AAV gene delivery was the host immune response, as the antibody against wide-type AAV is very common in the general population althoug it is non-pathogenic. As the AAV is one type of the single-strand DNA virus, the target gene expression usually need two to four weeks, so it may be an urgent problem for some acute conditions,such as stroke. The Mesenchymal stem cells(MSCs) are widely investigated for gene therapy, in vitro and in vivo evidence has suggested that MSCs are not intrinsically immunogenic, do not stimulate alloreactivity, and exert suppressive effects on T-cell proliferation. Objective:This study was desinged to observe that mesenchymal stem cells as carrier of the target gene in the brain transplant, the possibility of escape from the immune response against wild-type AAV in sensitized rats, in order to achieve the timely expression of target genes.Our preliminary study found that gene-modified MSCs by rAAV2-eGFP could evade the specific immune response of the host rats against the wide-type AAV2and effectively tranduce eGFP into brain by a allotransplant, and a stable expression of the target gene was detected.Methods:Rat bone marrow-derived mesenchymal stem cells modified by AAV2-eGFP in vitro and stablly expressing of eGFP were transplanted into the rats’brains that had been immunuologically sensitized by wild-type AAV. Immunofluorescence staining, Western Blot, and other methods were used to detect the cells survival and the eGFP expression.Results:Mesenchymal stem cells could be effectivelly transfected by AAV2-eGFP, and stable express eGFP in vitro. Repeated subcutaneous injection of wild-type AAV could make the rats infected with AAV, and produce high titers of neutralizing antibodies. The Mesenchymal stem cells transplanted into rat brain can survive and contiuned to express eGFP. No evidence of specific immune response was found. Conclusion:Our preliminary study found that gene-modified MSCs by rAAV2-eGFP could evade the specific immune response of the host rats against the wide-type AAV2and effectively tranduce eGFP into brain. And it provided a method of potential cilinical application to solve the problems of the immune response and delayed target gene expression, which hindered the development of gene therapy for cerebral ischemic diseases.