| Leukemia has become one of the most common hematopoietic malignancies in china with the incidence accounting top ten. and leukemia is the first most frequent cause of malignant disease-related death among young people. At present, patients with leukemia are greatly relieved as the result of comprehensive application of targeted therapy, hematopoietic stem cell transplantation and so on. But there are still a lot of problems such as chemotherapy-related toxicity, non-selectivity, drug resistance as well as relapsed or refractory leukemia. These questions prompt researchers to develop a more targeted and more powerful agents for fighting leukemia, and the gene-viral therapy is a important one among them. In recent years, there is a significant increase in the use of gene-viral therapy in clinical application. Viral vectors are by far the most popular vectors for gene delivery, adenoviruses being the most commonly used viruses (25%of clinical trials), Specifically,75%of adenoviral gene therapy trials are used for the treatment of cancer[2].Oncolytic adenoviruses is also called conditionally replicating adenoviruses (CRAd). It has become a promising strategy in cancer gene therapy largely due to its ability to selectively infect and replicate in tumor cells. In recent studies, It showed that Ad35tropism generated through the substitution of the Ad5fiber protein by the Ad35knob domain improved infectious efficiency and antitumor gene therapy effects of the Ad5tropism in leukemic cells These type of oncolytic adenoviruses were generated by deleting the viral E1B55KD gene, which resulted in not damaging normal cells, but deletion of E1B55KD reduces the replication and production of Ads, which in turn reduces the antitumor effrcacy[21]. So, It’s still urgent to develop a more potent and more targeted biologic agent.In this study, we constructed a novel chimeric Ad5f11pTPEGFP. It has dural characters:first, it has Ad11p tropism generated through the substitution of the Ad5fiber knob by the Adllp knob domain, thus possesses more higher infectious efficiency in infection of leukimia cells; second, it’s endogenous E1A promoter is substituted by a tumor-specific promoter TERTp, thus could restrict adenoviral replication to telomerase-positive tumor cells and efficiently lyse tumor cells, which make the chimeric Ad being more targeted. We proved in vitro that the chimeric Ad could infect leukemia cells, kill leukemia cells effectively. Our data suggest that this novel chimeric adenovirus agent has potential to be a useful vector vehicle for treatment of leukemia.In addition, In recent studies, it showed that the presence of either adenovirus or certain of its gene products supported the replication of B19V DNA in nonpermissive cells. Furthermore, B19V DNA replication can produces low level of progeny virus in nonpermissive293. But the helper function is weak in B19semipermissive cell line UT7/Epo-S1(a subclone of UT7/Epo) either infection with adenovirus or transfection with a subset of adenovirus genes. There is two reasons we considered may lead to this result:one is low infection efficiency of adenovirus in UT7/Epo-S1, the other is the difficulty in the transfection of plasmid into suspension cultures UT7/Epo-S1, The above reasons may influence expressing of adenoviral gene products. In this study, we observed replication of B19in UT7/Epo by the chimeric Ad infection, and we try to provide a support for further improving and making Ad a vehicle to rescue the progeny B19.This study is divided into two parts:Part Ⅰ:in vitro infection of chimeric adenoviruses in human leukemia cellsPart Ⅱ:observation of the assistance of chimeric adenovirus to the replication of human parvovirus B19 Part I:In vitro infection of chimeric Adenoviruses in human Leukemia cellsObjective To construct a recombinant adenovirus (rAd) Ad5f11pTPEGFP with a chimeric Ad5/fllp fibers, whose endogenous El A promoter is substituted by a tumor-specific promoter TERTp. We further confirm its antileukemia efficacy in vitro and try to provide a vector vehicle target therapy for leukemia. Methods An adenoviral plasmid pAd5f11pTPEGFP was constructed and rescued from pAd5f11pTPEGFP-transfected HEK293cells. The virus was purified by double CsC1density gradient centrifugation, and titrated with limiting dilution assay on HEK293cells. The gene transduction efficiency (since Ad5f11pTPEGFP carried green fluorescence protein reporter gene) and replication-competent ability of this chimeric Adenoviruses in human leukemia cell lines were evaluated through inverted fluorescence microscope, fluorescence-activated cell sorting assay (FACS) and viral titer testing; cytotoxicity on human leukemia cell lines of this chimeric Adenoviruses was determined by CCK-8; Leukemia cell apoptosis induced by Ad5f11pTPEGFP was determined by AnnexinV-APC/7-AAD and further proven by the level of cleavage of PARP, a molecular mark of apoptosis in early phage detected by western blot. Results (a) A novel recombinant Ad was successfully rescued and amplified, and the titer of the purified viruses was4.5X1010IU/ml (infection unit per milliliter). The recombinant Ad was significantly more effective than control Ad5GFP in infection on leukemia cell lines in vitro with time-dependent and dose-dependent manners, and also produced more significant higher level viral titer than Ad5GFP while infect with the same dose;(b) In contrast to Ad5GFP, the new recombinant Ad caused more significant cytotoxicity on leukemia cell lines;(c) The apoptosis rate of leukemia cells induced by Ad5f11pTPEGFP was significantly higer than that of Ad5GFP; the cleavage of PARP enzyme increased with time. Conclusion The chimeric Ad with both f11p and TPE gene was successfully constructed and prepared, which could infect leukemia cells, kill leukemia cells effectively, and induce apoptosis of the leukemia cells. Our data suggest that this novel chimeric adenovirus agent has potential to be a useful vector vehicle for treatment of leukemia. Part II:Observation of the assistance of chimeric Adenovirus to the replication of human parvovirus B19Objective To generate full length B19genome and to rescue progeny viruses B19by infection with chimeric Adenovirus in UT-7/Epo cells. Methods The pB19N105plasimid which contain full length B19genome with long ITRs was constructed by using low incubation temperatures and Sure2bacterial cells. UT-7/Epo cells were transfected with plasmids pB19N105using Nucleofector with the help of the recombinant adenovirus Ad5f11pTPEGFP. The cells were harvested at various times post-transfection, the copies of B19genome were monitored by Real-time PCR during different time points(0h,24h,48h and72h); cutting1/2ITR off from both terminal of full length B19genome, a plasmid carrying B19genome with1/2ITR was constructed, it was then coloned to a pShuttle plasmid and recombinated with pAdEasy/llp in E.coli strain BJ5183to generate recombinant Ad5plasmid. This recombinant Ad5plasimid with B19genome was transfected into HEK293to rescue the pAd5f11p-h1B19. The copies of recombinant Ad5genome and B19genome were monitored by Real-time PCR during different time points. Results pMD-B19N105which contain the full length B19genome was successfully constructed, the copies of B19genome didn’t increase with time while pMD-B19N105was transfected into UT-7/Epo cells by the chimeric adenovirus infection; The recombinant plasmid pAd5f11p-h1B19which contain1/2ITR of B19genome was successfully constructed; The copies of Ad5genome didn’t increase with time and the copies of B19genome decreased with time dramatically during transfection of pAd5f11p-hB19into HEK293. Conclusion The full length B19genome with long ITRs was successfully cloned. Progeny virus B19wasn’t rescued from pMD-B19N105by infection with chimeric adenovirus; The replication of recombinant Ad5fllp-h1B19was hampered and B19genome was lost while pAd5fllp-h1B19was transfected into HEK293. Our research provide a support for further improving and making Ad a vehicle to rescue the progeny B19. |
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