Gene Therapy With Novel Adenovirus Vector Specifically Targeting HCC

In our studies, we constructed series of conditionally replicative adenovirus (CRAd).Genes from the early region (E1) in adenovirus 5 were deleted and replaced with modifiedα-fetoprotein (AFP) promoter. The AFP promoter contains several silencer and enhancerelements, which are responsible for silencing the promoter in adult liver cells andenhancing the activity of the promoter in fetal liver cells. We inserted a regulatory element,HS-4 insulator, in the upstream of AFP promoter and inserted E1A gene, reporter gene AP,or therapeutic gene TRAIL in the downstream of AFP promoter. Expression of foreigngene inserted into CRAds is under the control of AFP promoter. Through constructingseries of recombinant adenoviruses controlled by AFP promoter, we study the function ofE1A gene in CRAds and CRAds specific killing efficiency to AFP-expressing HCC celllines. We also research the influence of HS-4 insulator to the specificity and transcriptionactivity of foreign promoter. Furthermore, we study cancer therapeutic efficacy ofcancer-specific CRAds that express therapeutic gene.We constructed vectors Ad.IR-AP and Ad.IR-AP/E1A to study E1A function in CRAds. TheIn vitro experiments indicated that the E1A expression increased replicative efficiency ofAd.IR-AP/E1A and produced stronger cytopathic effect (CPE) induced by Ad.IR-AP/E1Aas compared with Ad.IR-AP in cancer cell lines. In vivo, Immunodeficient mice withhepatic metastases were injected with Ad.IR-AP/E1A via tail-vein. Results showed that theE1A expression effectively improved the replication and spread of CRAds in cancer cells.We then constructed vectors Ad.AFP-AP and Ad.HS4.AFP-AP to study target geneexpression of adenovirus under AFP promoter control in AFP-expressing primary HCCcells. On the other hand, we studied the specific regulation to AFP promoter by regulatoryelement, HS-4 insulator. Results showed, in AFP-expressing HCC cell lines, thatAd.AFP-AP specifically expressed reporter gene AP under the control of AFP promoter.Whereas Ad.HS4.AFP-AP, which was inserted HS-4 insulator in front of AFP promoter,increased HCC-specificity and the activity of AFP promoter in vitro and in vivo.Based on the results of studies above, we constructed a clinical potential CRAd,Ad.HS4.AFP.E1A/TRAIL, which can specifically express therapeutic gene TRAIL inAFP-expressing HCC cell lines to improve its HCC killing specificity and efficiency. The 6study demonstrated that Ad.HS4.AFP.E1A/TRAIL had tight AFP-expressing HCC killingspecificity and can effectively kill HCC cells in vitro and in vivo.This study explored a kind of CRAds that was controlled by tumor-specific heterologouspromoter and regulatory element HS-4 insulator. This CRAd can replicate and expresstherapeutic gene in target tissue, which provide a new strategy for cancer gene therapy.