Preliminary Evaluation of Safety of Conditionally Replication Adenovirus M4

Conditionally replication adenovirus M4, which was constructed in our lab, was proved to have good clinical application prospect for its good antitumor and antimetastasis effect. However, clinically applying M4 faces many problems. One of the most important is the safety of M4. In this study, we investigated the safety of M4 by comparing with Adv-TK, which was proved to be safe in Ⅰ–Ⅲ phase clinical trials. M4 and Adv-TK were injected into mice via the tail vein separately, and the mice were sacrificed at the indicated time. Blood was collected for biochemical tests, the liver was harvested for hematoxylin and eosin (H&E) staining and viral quantification, and splenic lymphocytes were separated for adenovirus specific cellular immune response. Our results showed that M4 had no obvious effect on mouse general symptoms. A transient reversible infiltration of inflammatory cells in collect abbacy was only observed in M4 group, and a transient slight increase in Cr level was detected both after M4 and Adv-TK injection. The adenovirus specific cellular immune response induced by M4 was similar to that by Adv-TK, and the distribution and metabolism of M4 in the mouse liver were also similar to those of Adv-TK. It was concluded that conditionally replication adenovirus M4 had the same safety as Adv-TK. The study provides safety basis for the coming clinical trials of M4.

An Oncolytic Adenovirus Expressing Herpes Simplex Virus-Thymidine Kinase for Targeting Cancer Therapy:An in vitro Evaluation

Objective： Oncolytic adenovirus, also called conditionally replicating adenovirus （CRAD）, has been developed for the treatment of cancer. However, there is a tremendous need to enhance their antitumor efficacy. Here we wish to evaluate whether a strategy that combines the herpes simplex virus-thymidine kinase with oncolytic effects offers a therapeutic advantage. Methods： A novel adenovirus Ad-ETK containing a sequentially positioned promoter of human telomerase reverse transcriptase （hTERT）, the coding sequence of E1A gene, an internal ribosome entry site sequence （IRES） and the coding sequence of herpes simplex virus-thymidine kinase （HSV-TK） was constructed. Infection of various cells with Ad-ETK followed by RT-PCR confirmed the expression of E1A and HSV-TK. The oncolytic ability and synergism between oncolytic effects and HSV-TK system was measured. The infection efficiency was determined by flow cytometry. Results： Ad-ETK deliverys E1A and HSV-TK gene, which selectively replicates in hTERT-positive tumor cells, and the progeny virus can reach up to 150 IU/cell. Our in vitro study showed that Ad-ETK plus ganciclovir （GCV） induced an obvious cell death. Conclusion： An oncolytic adenovirus plus the HSV-TK/GCV suicide gene system resulted in a significant improvement in treatment efficacy and it may offer important considerations in the development and preclinical assessments of oncolytic virotherapy.

PREPARATION OF GENE-VIRAL THERAPEUTIC SYSTEM CNHK200-HA AND ITS ANTITUMOR ACTIVITY ON LUNG CANCER

Objective： To develop a novel adenoviral vector system, which combines the advantages of the antiangiogenic gene therapy and virus therapy, and to investigate its antitumor activity on lung cancer. Methods： A new kind of viral vector CNHK200, in which the Elb55kDa gene was deleted and the whole Ela gene was preserved, was constructed. Human angiostatin gene Kringle 1-5 （hA） was amplified and inserted into the genome of the replicative virus CNHK200, generating CNHK200-hA. The expression of hA and its effect on lung cancer cell growth in vitro and in vivo were studied. Results： The novel vector system CNHK200-hA, just like the replicative virus ONYX-015, replicated in p53-deficient tumor cells but not in normal cells, and thus specifically killed tumor cells. In in vitro experiment, both CNHK200-hA and the non-replicative virus Ad-hA could kill tumor cells but the latter needed 100 times more MOI to achieve the same level of cell killing. In in vivo experiment, the therapeutic effect of CNHK200-hA on human lung cancer A549 xenografts in nude mice was significantly better than that of Ad-hA or that of ONYX-015. Conclusion： CNHK200-hA, which carries the angiostatin gene, has the advantages of specific tumor targeting, high expression of transgene in tumor cells and potent antitumor activity.

Anterograde Trans-Synaptic Tagging Mediated by Adeno-Associated Virus

Trans-neuronal viral tracing is becoming one of the most important methods for mapping neuronal circuit connections,but an anterograde trans-synaptic tracer compatible with functional assays is still lacking.Adeno-associated virus（AAV）is replication-defective,and its production of offspring needs the help of adenovirus（AV）[1]or herpesvirus co-infection[2].The replication deficiency,non-pathogenicity,easy manipulability,