Therapeutic RNA Silencing Of CX₃CL1 In Viral Hepatitis And Cancer

In September 1999,the perceptions of the use of adenoviral(Ad)vectors for gene therapy were altered when a patient exposed via the hepatic artery to a high dose of adenoviral vector succumbed to the toxicity related to vector administration. Appropriately,concerns were raised about continued use of the Ad vector system and, importantly,there were increased efforts to more fully understand the toxicity.Today it is recognized that there is no ideal vector system,and that while Ad vectors are not suitable for all applications,the significant advantages over other vector systems including efficient transduction of a variety of cell types,both quiescent and dividing, make it optimal for certain applications.These include protocols where high levels of short-term expression are sufficient to provide a therapeutic benefit.Potential target applications include therapeutic angiogenesis,administration into immune-privileged sites such as the CNS,or treatments where the adjuvant effect of adenovirus can be of benefit such as cancer vaccines.Broader applicability of Ad vectors will require resolution of toxicity issues.The researches presented here focus on the adenovirus-induced acute liver injury,induction of in vivo hepatic immuno suppression against adenoviral infection and gene therapy of melanoma.1,Hydrodynamic injection of adenovirus to construct a novel liver injury model.So far,it has been widely reported that adenovirus vectors delivered by i.v. injection accumulate in the liver and induce liver injury.But few common animal models were established to study the adenovirus-induced liver injury.The purpose of our research is to construct a novel adenovirus-indued fulminant hepatitis model to study the mechanisms involved in the adenovirus-induced auto-immune hepatitis.We employed hydrodynamics-based injection(10%body weight and the tail vein injection was performed over less than 5 s)to deliver adenovirus vector into the mouse livers and observe the pathological changes after infection.C57 BL/6 mouse was hydrodynamic injection with different adenovirus vectors(AdLacZ,AdEGFP and AdsiNeg),and then assayed the proportion and total amount of hepatic lymphocytes by FACS.The serum ALT levels and pathological H&E staining of liver sections were used as indication of liver injury.We found that hydrodynamic injection of adenovirus vector induced much more severe liver inflammation than conventional i.v. injection.HP-injection of adenovirus preferentially led a substantial increase of lymphocytes in infected liver.Especially the hepatic NK cells were significantly increased and activated.It was also observed that the serum ALT levels were dramatically increased following severe liver necrosis.The transcript level in infected liver and the serum level of IFN-γwere both up-regulated.Depletion of NK/NKT cells or NK cells prevented mice from adenovirus vector-induced liver injury and down-regulate the expression of IFN-γ.All these data indicated that adenovirus infection induced the infiltration and activation of hepatic NK cells leading to liver injury and inflammation.So NK cells are the key effector cell in the early phase of adenovirus infection in this model.In conclusion,our work presents a novel viral hepatitis model associated with human acute liver viral infection and provides a efficient platform for studying adenovirus infection.2,Therapeutic RNA silencing of CX3CL1 prevent adenovirus induced liver injuryWe investigate the role of chemokine CX3CL1/fractalkine(FKN)involved in the anti-viral infection immunity.Fractalkine,the only member of CX3C chemokine containing both membrane-anchored and soluble forms,has both chemoattractant and cell-adhesive functions and is believed to be an important regulator of inflammatory response as an inducer of cellular infiltration,including induction of IFN-γ.Most of NK cells,CD14~+ monocytes and partial CD8~+ T cells express its receptor CX3CR1. By further studying the hydrodynamic infection of adenovirus induced liver injury model,we found that the expression of CX3CL1 in adenovirus infected liver was significantly upregulated,and interestingly,the CX3CR1 expression on the infiltrated NK cells was also up-regulated.To explore the relationship between them,we constructed adenovirus vector to express mouse recombinant CX3CL1(AdFKN). Injection of AdFKN induced the overexpression of CX3CL1 in mouse liver and dramatically promoted the infiltration and activation of CX3CR1~+ NK cells while the serum ALT levels and the liver necrosis were increased too.These results indicated that adenovirus infection induced the expression of hepatic CX3CL1,then NK cells were recruited and activated through the interaction between CX3CL1 and CX3CR1 following the production of IFN-γ.So,inhibition of CX3CL1 expression might be a good choice for eluding adenovirus vector immunity.We constructed adenovirus vector carrying mouse CX3CL1 specifc siRNA(AdsiFKN)to perform in vivo RNA interference.After hydrodynamic injection of AdsiFKN,the expression of CX3CL1 in infected mouse liver was greatly inhibited.At the meantime,the infiltration of NK cells and the liver inflammation were significantly down-regulated too.In order to further demonstrating the pro-inflammatory role of CX3CL1,the mice were pre-treated with anti-CX3CL1 or anti-CX3CR1 specific neutralizing antibody before virus injection.We found that the injury and inflammation of infected liver could be greatly suppressed too.Our findings suggest a strategy to prevent or alleviate adenovirus vector-induced acute liver injury by blocking CX3CL1/CX3CR1 interaction in adenovirus vector-based gene therapy.3,Down-regulation of surface fractalkine by RNA interference in B16 melanoma reduced tumor growth in miceIt is reported that membrane-bound form of CX3CL1/fractalkine(FKN)promotes cell-cell adhesion by binding with its unique receptor CX3CR1,and membrane fraetalkine works as an adhesion molecule for the communication between tumor cells and vascular endothelial cells.Here,we show that CX3CL1/fractalkine was expressed on both mouse and human solid tumors,and small interfering RNA-mediated knock down of fractalkine gene inhibited melanoma B16-F0 cells growth in vivo,which was correlated to the decreased angiogenesis around the tumor. Our findings for the first time suggest that membrane fractalkine may possibly promote tumor angiogenesis through its strong cell adhesion function and therefore serves as a potential target of tumors therapy including RNA interference.