| Tumor is a disease of a serious threat to human health.Although classical cancer therapies were widely applied in cancer therapy,they caused obvious side-effects in normal cells because they are lack of tumor specificity.Genetically engineering oncolytic adenovirus can be only amplified in tumor cells,namely conditionally.replicating adenovirus(CRAd).The optimized and improved oncolytic adenovirus has been applied in clinical treatment,but it still has some shortcomings,such as high immunogenicity,liver toxicity,the limited infection area and the existence of the security risk of adenovirus replication when the virus was administrated by an intratumor injection.One way to deal with the above issues is that the oncolytic adenovirus for gene therapy in vivo was delivered using Mesenchymal stem cell(MSC).MSC are pluripotent stem cells were originated from non-hematopoietic cells.They can be collected from multiple types of tissues,such as bone marrow,adipose tissue,peripheral blood,umbilical cord blood,bone tissue,muscle and cartilages.MSCs have been widely used to deliver therapeutic gene and virus vector in cancer gene therapy due to their homing ability and immune evasion.When MSCs were used for a delivery vector for oncolytic adenovirus,the function of MSCs,such as homing and migration,is reduced due to the replication of virus in MSCs,which results in the attenuation of virus delivery and thus decreases gene therapy efficacy.To efficiently deliver oncolytic adenovirus by MSCs,it is necessary to maximize the amount of virus loading without compromising MSCs function(homing and migration).To solve this problem,a control system for virus replication is a promising approach.This system will not allow for virus gene replication until MSCs arrive at cancer tissue,which will make it possible that maximal virus loading in MSCs will be achieved without affecting the function of MSCs.In addition,this system will bring a safer way to deliver virus into specific location of cancer tissue.Overall,our control system will bring a huge beneficial impact to gene therapy.One way to achieve this goal is to use a well-known gene expression regulation,Tet,which can manipulate the expression of genes efficiently and safely.E1A gene regulates the initiation of gene replication in adenovirus.Constructing Tet-on system for controlling E1A gene expression will allow us to turn on virus genome replication after MSCs arrive at cancer tissues.Three specific aims are addressed as the following in the project.1.Construction and detection of conditional replicating adenovirus with its replication controlled by rtTA mediated Tet-on regulation systems:Three types of rtTA mediated Tet-on systems for controlling E1A expression and their corresponding viral vectors were established.Results showed that all three systems failed to control virus replication.To clarify its reason,luciferase reporter gene was used for analyzing the problems and the results indicated that the efficiency of Tet-on system was compromised by E1A gene products,which resulted in the failure to control the replication of virus.Therefore,the rtTA mediated Tet-on system is not an optimal approach in controlling virus replication.2.Construction and detection of conditional replicating adenovirus with its replication controlled by TetR-KRAB mediated transcriptional inhibitory Tet-on regulation system in human MSCs(hMSCs):After constructing TetR-KRAB mediated transcriptional inhibitory Tet-on regulation system and its corresponding CRAd vector,the virus replication efficacy of the oncolytic viruses and hMSC loading capacity were evaluated in vitro.The results showed that this system can efficiently control virus replication in both cancer cells and hMSCs.Meanwhile,the amount of loading virus in hMSCs was significant increased by one order of magnitude.3.The experimental study of hMSC loaded with CRAd with its replication controlled by TetR-KRAB mediated transcriptional inhibitory Tet-on regulation system in breast cancer mouse model in vivo:The results showed that hMSC loaded with the novel virus can significantly inhibit the growth of cancer compared with the control groups,and the inhibitory effect of the viruses on tumor growth could be regulated by Dox.In summary,we clarified the reasons that rtTA mediated Tet-on regulation systems is not applicable for regulating adenovirus gene replication.Then we constructed TetR-KRAB mediated transcriptional inhibitory Tet-on regulation system,which can efficiently control adenovirus gene replication by regulating E1A gene expression.In addition,the loading capacity of viruses in hMSCs was significantly increased,which guaranteed that TetR-KRAB mediated transcriptional inhibitory Tet-on regulation system can work well in hMSCs.More importantly,our in vivo results showed that CRAd with this system can efficiently suppress breast cancer in mice,which can be manipulated by Dox.This study is innovative and would significantly contribute to cancer gene therapy. |
PDF Full Text Request