Evaluation Of Anti-tumor Therapeutic Effects Of Oncolytic Adenoviral Vectors In Gene Therapy And Combined Therapy With Chemotherapeutic Agents

Cancer is a group of heterogeneous diseases,results due to uncontrolled cell division.Several genetic and environmental factors contribute to developing cancer.Cancer remained a significant health problem and one of the foremost causes of diseaserelated deaths in the world.Cancer metastasis and variable treatment responses to anticancer regimens in cancer patients are major hurdles in cancer treatment.Chemotherapy remained the popular choice to manage cancer for the last several decades.Chemotherapeutic agents,synthetic and originated from plants,exhibited fruitful anti-cancer outcomes in cancer patients.However,persistent usage of chemotherapeutic agents found out to be associated with issues like the development of adverse effects and drug resistance.Therefore,it is urgently needed to develop the nontoxic anti-cancer agents and to implement these anti-cancer agents in treatment strategies,primarily to manage the metastatic stage of the disease.These non-toxic anticancer agents can be investigated in monotherapy as well as combined therapy with chemotherapeutic agents against cancer.Adenoviral vectors have long been used for gene therapy in humans.Adenoviral vectors were the first viral-based vectors used in humans for gene therapy.These considered being an attractive option due to harboring merits like high transduction efficiency,comparatively low toxicity,and cost-effective production at a large scale.In different studies conducted to examine the effectiveness of adenoviral-based therapies,mainly two different types of adenoviral vectors are employed.One type is oncolytic conditionally replicating adenoviral vectors,and the other is replication-defective adenoviral vectors.Oncolytic viruses can selectively infect and lyse tumor cells and offer an attractive anti-cancer avenue.Adenoviruses are among the most frequently employed agents for the delivery of tumor suppressor genes or to induce direct oncolysis in tumor cells.The use of a conditionally replicating adenovirus(CRAd)to treat head and neck cancer in China,and very recent acceptance of an oncolytic virus-based treatment in the United States raises optimism on the anti-cancer potential of oncolytic virotherapy.Therapeutic replication-defective adenoviral vectors tend to deliver genes of interest at the tumor site.These genes in-turn,can perform their tumor inhibition function.Replication-defective adenoviral vectors can be constructed by deleting the E1A/B region of the adenovirus genome.E1A/B genes are required for the replication of adenoviruses.Deletion of E1A/B genes will not only make the replication of the viral vector deficient as well as provide the opportunity to insert genes of therapeutic nature like p53 and p14 ARF tumor suppressor genes.Several studies have reported the potent anti-tumor efficiency of such adenoviral vectors.P53 is a well-known and well-studied tumor suppressor gene.In response to DNA damage and cellular stress signals,p53 is activated and triggers a myriad of downstream events responsible for tumor inhibition.The profound tumor inhibitory effects of the p53 pathway include apoptosis induction,cell cycle arrest(G1-S,G2-M),inhibition of angiogenesis,and DNA repair.The deficiency or mutation in the p53 gene is present in > 50% of cancers.The delivery of the p53 gene through viral vectors will restore the P53 protein level in p53 non-expressing tumors but also raise the level of p53 protein in p53 expressing tumors.In both scenarios,exogenous p53 will ultimately suppress tumor growth.P14 ARF is another tumor suppressor gene.It also has a role in stabilizing the P53 level by interfering with p53 inhibitor,MDM2 ubiquitin ligase.In this way,it helps p53 to perform its function uninterrupted.The cloning of these potent tumor suppressor genes in adenoviral vectors and their delivery to tumor sites may circumvent tumor progression and achieve efficient tumor inhibition outcomes.Although adenoviral vectors can show potent anti-tumor effects,one major obstacle lies in their usage in the clinical setting is their delivery to tumor sites.Intratumoral injections of adenoviral vectors suppressed the tumor growth in many studies,but their systemic delivery could not produce satisfactory results.Possible reasons for such low practical effectiveness include high doses of viral vectors(> 1012 particles)were required,most of the virus particles fail to disseminate into the tumors and removed by the reticuloendothelial system,and the activation of the host immune system in response to the viral vectors.These hurdles can be avoided by developing new approaches to deliver adenoviral vectors systemically.Mesenchymal stem cells(MSCs)could prove efficient delivery vehicles for adenoviral vectors.MSCs possess particular advantages like tumor tropism and tumor homing ability.Moreover,these can evade the immune response and disseminate into the tumor mass.Certain modifications in MSCs may enable these to package,propagate,and deliver adenoviral vectors specifically to the tumor site.These MSCs based vehicles will target not only the localized tumors but also metastatic tumors.Given aforesaid merits and literature-based evidence,we developed a novel MSCsbased delivery system for adenoviral vectors.We made modifications to MSCs by equipping these with adenovirus E1A/B genes and constructed MSC-E1 s.E1A/B genes are essential for adenoviral replication.We investigated the tumor tropism and homing ability of MSC-E1 s after systemic delivery of MSC-E1 s to tumors in mice models.Biodistribution analysis of MSC-E1 s and intravital microscopy of tumors in mice models revealed successful tumor tropism and intratumoral dissemination of MSC-E1 s.Our research group constructed replication defective adenoviral vectors expressing p14ARF/P53(Ad-bicistronic or Adbic)and Survivin regulated conditionally replicating adenoviral vector(CRAd).We tested and compared the anti-tumor potency of free and adenoviral vectors(CRAds and Adbic)loaded MSCs in prostate tumor cells,in-vitro,and in-vivo.Results of cytotoxicity and apoptosis assays showed that tumor cell inhibition was more prominent in MSC loaded adenoviral vectors treated cells.Similarly,tumor growth inhibition was more prominent in prostate tumor mice models received systemic delivery of adenoviral vectors loaded MSC-E1 s than the free adenoviral vectors.Additionally,we also evaluated the tumor inhibition efficiency of adenoviral vectors in combination with Cisplatin and Irinotecan.Prostate tumor volume was greatly reduced in the combined treatment group than the mono treatment group.CRAds treatement on chemo-sensitive and resistant lung cancer cells successfully inhibited the proliferation of cancer cells.The chemo-resistant cancer cells exhibited high expression of coxsackievirus and adenovirus receptor(CAR),as it was reported to interact with viral capsid proteins resulting in its internalization.we carried out CAR knockdown using CAR-si RNA and CAR expression using CAR c DNA in chemosensitive and resistant cells,respectively.CAR protein analysis through western blotting shows that both experiments went successful.Furthermore,X-gaining showed that compared to controls,adenoviral transduction and tumor inhibition was siginificantly reduced in cancer cells which received CAR-si RNA.On the contrary,cells which received CAR c DNA exhibit higher viral infection and subsequent tumor cell death compared to the control group.We concluded cisplatin-driven upregulation of coxsackievirus and adenovirus receptor(CAR)as a selective vulnerability of chemotherapy-sensitive and resistant cancers.We investigated the tumor suppression activity of Adbic loaded MSC-E1 s alone and in combination with Aloperine(ALO),an herbal alkaloid,against non-small cell lung cancer(NSCLC).Moreover,the anti-tumor efficacy of Adbic loaded MSC-E1 s alone and in combination with Apigenin(API)was assessed in Pancreatic cancer(PC).Results of invitro experimentations(MTT Assay,Flow cytometry-based apoptosis analysis,DAPI staining,TUNNEL assay,western blotting analysis)revealed that Adbic loaded MSC-E1 s alone and in combination with Aloperine and apigenin synergistically inhibited the growth of NSCLC and PC,respectively.We found out that a possible mechanism for this finding is the activation of the p53 pathway.The western blotting analysis showed increased levels of p53 proteins in tumor cells treated with Adbic loaded MSC-E1 s alone or in combination with chemotherapeutic agents compared to control cells.P53 triggered downstream events lead to the achievement of anti-proliferative activities like apoptosis induction,cell cycle arrest(G1-S in NSCLC,and G2-M in PC),upregulation of pro-apoptotic(Bax,Caspase 3 and Caspase 9)and downregulation of anti-apoptotic gene(Bcl2).Similarly,results of in-vivo studies also exhibited that delivery of Adbic loaded MSC-E1 s alone or in combination with ALO and API to tumor mice models inhibited the growth of NSCLC tumors and PC tumors,respectively.Tumor growth significantly reduced in combination treatment groups.In our current study,we constructed adenovirus type 5 E1s-modified MSCs based vehicle system(MSC-E1s)for the systemic delivery of adenoviruses to the tumor site.Investigations revealed that MSC-E1 s are well capable of executing the packaging,propagation,tumor targeting,and tumor homing activities.Adenoviral vectors loaded MSC-E1 s inhibited the proliferation of prostate,NSCLC,and pancreatic cancer cells substantially compared to free Adenoviral vectors.Adenoviral vectors loaded MSCE1 s administered to subcutaneous tumor models via tail vein(systemic delivery).These successfully targeted the tumors,showed excellent intratumoral dissemination,and suppressed the tumor growth in mice models compared to freely delivered adenoviral vectors.In combined treatment approaches,adenoviral vectors in combination with chemotherapy drugs produced synergistic tumor inhibitory effects.Combined treatment mediated activation of the p53 pathway observed to be the most probable reason for achieving synergistic inhibitory effects.Combined treatments prolonged the survival rate in NSCLC mice models and suppressed the prostate and pancreatic tumors growth in mice models.So,these productive findings encourage the investigation of these strategies at the advanced levels,and these may offer the possibility of devising new treatment approaches to combat cancer.