Construction And Application Of A Novel Oncolytic Adenovector System

Oncolytic adenovectors, also called conditionally-replicating adenoviruses (CRADs), have been widely applied in cancer gene therapy. CRADs can selectively propagate in and then lyse tumor cells, and the released progeny viruses can again infect the neighboring cancer cells. As the cascade response proceeds, tumor is destroyed in the end. The viral replication process stops when CRADs reach and infect normal cells, therefore, it is safe for normal cells. Transgene was transferred to tumor cells by replication-defective adenovector in traditional cancer gene therapy, However, the clinical outcome is discouraging due to low gene transfer efficiency. CRADs have the advantage over replication defective viruses in spreading of progeny.Traditionally, the CRADs were rescued by homologous recombination of shuttle and backbone plasmids in 293 cells. The recombination in eukaryotic cells is low-efficiency and time-consuming. Moreover, plaque purification was indispensable to isolate the correct recombinant. The situation of inefficient adenoviral construction was changed since the establishment of AdEasy system in 1998. In this system, recombination between shuttle and backbone plasmids occurs in prokaryotic cells, which is more efficient. Furthermore, plaque purification is unnecessary for the recombinant adenovirial plasmid is homogeneous as extracted from a single bacterial colony. Originally, the AdEasy system was used to construct the replication-defective Ad vectors. Based on AdEasy system, a simplified system for generating oncolytic adenovirus vector carrying one or two transgenes was established in this study. The Oncolytic effects of the generated CRADs were also investigated in vitro and in vivo.A novel plasmid, pTE-TPE-GM, was constructed. pTE-TPE-GM has three characteristics: firstly it carries Ad5 E1 genes, which can make the generated CRAD to replicate selectively in tumor cells; secondly, it contains a small genome so that modification of E1 gene can be operated conveniently; finally, the modified E1 gene can be easily excised from this plasmid and subcloned into the proper site of Shuttle plasmid. In the novel plasmid, E1A promoter was replaced by tissue-specific promoter (TSP) and E1B19K coding sequence was replaced by the first transgene GM-CSF. The internal ribosome entry site (IRES) sequence was inserted between the transgene and E1B55K to ensure the expression of E1B55K. The modified E1 region was precisely integrated in the joint point between SV40 ployA signal and the right homologous arm of pShuttle-CMV to obtain a novel shuttle plasmid, which was used to cotransform the E. coli BJ5183 with AdEasy-1 plasmid. After homologous recombination, an adenoviral plasmid carrying one transgene could be generated, which can be used to produce CRAD by transfecting 293 cells. If the second transgene was inserted into the multiply clone site (MCS) of pShuttle-CMV before insertion of the modified E1, a CRAD carrying two transgenes can be generated similarly. After the construction of pTE-TPE-GM, in which the telomerase reverse transcriptase promoter (TERTp) was selected to control the expression of E1A gene, two oncolytic adenovectors were designed and constructed based on this system. The first one is named as Ad-CD80-TPE-GM, which carried two transgenes of GM-CSF and B7-1 (CD80), and the other one is Ad-GFP-TPE-GM, which carried GFP and GM-CSF genes. Ad-GFP, a replication-defective adenovector, was used as negative control in this study.The oncolytic effect of Ad-CD80-TPE-GM and Ad-GFP-TPE-GM was examined in vitro. It was firstly observed that both of the CRADs could replicate in and kill PC3M cells. Furthermore, eight telomerase-positive and two telomerase-negative cell lines were selected to estimate the oncolytic effects of these viruses. The cell viability was measured by crystal violet staining at 7 days postinfection by the CRADs. These results showed that both Ad-CD80-TPE-GM and Ad-GFP-TPE-GM could selectively kill the telomerase-positive cells high-efficiently. When being infected by Ad-CD80-TPE-GM at an MOI of 10, nearly all the telomerase-positive cells were killed. About 80% cells were killed when infected at a dose of 1 MOI. Oncolytic effect of Ad-CD80-TPE-GM is also obvious when the value of MOI decreased to 0.1. On the contrary, neither CRADs showed effect on telomerase-negative cells. Ad-CD80-TPE-GM could replicate selectively in telomerase-positive Hep2 cells and produced about 373 infectious units (IU) progeny viruses in each cell.The expression of CD80 and GM-CSF in Ad-CD80-TPE-GM-infected cells was also investigated, using a replication-defective adenovector, Ad-p53/GM-CSF/B7-1, as a control. When Hep2 cells were infected by Ad-CD80-TPE-GM at an MOI of 5, GM-CSF concentration in the supernatant reached 100ng/ml 48h post infection, which is about 9000 times the amount of that in the control group. Similarly, the percent of CD80-positive cells was closed to 100%, and the expression intensity was about 146 times greater than the control group.The anti-tumor activity of Ad-CD80-TPE-GM was further investigated in the xenograft tumor model. Hep2 cells were implanted subcutaneously into the right flank of BALB/C nude mice. A tumor suppression rate of 78% could be observed after injection of Ad-CD80-TPE-GM with a dose of 1×10~9 IU. No increase of serum transaminase could be detected. The expression of CD80 and GM-CSF as well as viral replication could be detected at day 4 after the injection of Ad-CD80-TPE-GM. Intratumoral injection of Ad-CD80-TPE-GM could slightly induce neutralizing antibody against the viruses in nude mice.A simplified system for generating oncolytic adenovirus vector carrying one or two transgenes was constructed in this study. This system is flexible and can be modified to generate novel transcriptionally-regulated CRADs carrying other transgenes. Based on this system, Ad-CD80-TPE-GM carrying CD80 and GM-CSF was successfully constructed, and all data demonstrated a perfect anti-tumor effect of this CRAD. In conclusion, our work established a novel CRADs generation system for cancer gene therapy.