The Research Of Gene Therapy To B-lymphoma Using Sttrail Driven By A Specific CD20Promoter Mediated By Adenovirus Vector

CD20is a specific marker of B-lymphocyte, and the promoter of CD20has aspecific transcriptional activity in B-lymphocyte. Up to90%of non-Hodgkin’slymphoma （NHL） originates from B cells and have characters associated with asubpopulation of pre-B cells and a few mature B cells. Therefore, the CD20promoterhas been considered as a potential gene therapy for treating B-NHL. TRAIL is theonly one cytokine currently known as a selective killer to tumor cells. Furthermore,there is a ‘bystander’ effect in the gene therapy using TRAIL gene mediated byadenovirus vector to some cancers. In the present work, we chose to take CD20promoter as the targeted element; the product of stTRAIL gene as the cytotoxicprotein; adenovirus vector as the DNA delivery vehicle, and thus aimed at exploring anew strategy of gene therapy to treatment of B-NHL.Firstly, we cloned a477bp fragment of CD20promoter from the genome DNA ofBJAB, a CD20-positive cell line. And the specific activity of the CD20promoterfragment in CD20positive cell lines was measured. Secondly, an artificial TRAILgene sequence was constructed and the product of this sequence was termed asstTRAIL, which contained a secretion signal, an isoleucine zipper, and a TRAILfragment (114aa-281aa). The isoleucine zipper is a trimerization-enforcing domain ofstTRAIL, which facilitated the TRAIL fragment to form homotrimer and to initiateapoptosis. At last, an intact transcriptional unit which comprised with CD20promoterfragment, stTRAIL gene sequence and poly (A) sequence, has been inserted intoAdEasy adenovirus vector system. After packaged in293A cells, the virus termed asAdP20-stTRAIL was purified and concentrated into a high titer. Further experimentsconfirmed that the stTRAIL protein was expressed only in the CD20positive cellwhich was infected by AdP20-stTRAIL so that the stTRAIL protein could be secretedfrom the targeted cells. Most importantly, in vitro and in vivo experimentsdemonstrated the apoptotic cell death was induced in the infected BJAB cells by thestTRAIL protein secreted from the cells themselves.