Recombinant Oncolytic Adenovirus Research Articles

Treatment Response to Oncolytic Virus in Patient-Derived Breast Cancer and Hypopharyngeal Cancer Organoids: Evaluation via a Microfluidics Organ-on-a-Chip System

In this study, we present an oncolytic virus (OV) evaluation system established using microfluidic organ-on-a-chip (OOC) systems and patient-derived organoids (PDOs), which was used in the development of a novel oncolytic virus, AD4-GHPE. An OV offers advantages such as good targeting ability and minimal side effects, and it has achieved significant breakthroughs when combined with immunotherapy in recent clinical trials. The development of OVs has become an emerging research focus. PDOs can preserve the heterogeneity of in situ tumor tissues, whereas microfluidic OOC systems can automate and standardize various experimental procedures. These systems have been applied in cutting-edge drug screening and cell therapy experiments; however, their use in functionally complex oncolytic viruses remains to be explored. In this study, we constructed a novel recombinant oncolytic adenovirus, AD4-GHPE, and evaluated OOC systems and PDOs through various functional validations in hypopharyngeal and breast cancer organoids. The results confirmed that AD4-GHPE exhibits three antitumor mechanisms, namely, tumor-specific cytotoxicity, a reduction in programmed death ligand 1 (PD-L1) expression in tumor cells to increase CD8+ T-cell activity, and granulocyte–macrophage colony-stimulating factor (GM-CSF) secretion. The evaluation system combining OOC systems and PDOs was efficient and reliable, providing personalized OV treatment recommendations for patients and offering industrialized and standardized research ideas for the development of OVs.

Apolipoprotein A1-encoding recombinant adenovirus remodels cholesterol metabolism in tumors and the tumor microenvironment to inhibit hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a prevalent malignant tumor requiring effective treatments. Oncolytic viruses induce anti-tumor responses but have limited efficacy. Apolipoprotein A1 (ApoA1) inhibits inflammation, modulates immunity, and promotes anti-oxidation. This study aims to construct an oncolytic adenovirus (Ad5)-ApoA1 for superior anti-tumor effects. We analyzed ApoA1 expression in tumors and its prognostic significance using public databases. Subsequently, we engineered a recombinant oncolytic adenovirus Ad5-ApoA1 and assessed its replication and oncolytic efficacy in vitro and in nude mice. The impact of Ad5-ApoA1 on the tumor microenvironment of HCC was evaluated through flow cytometry, transcriptome sequencing, single-cell sequencing, and other methodologies. Additionally, mechanisms of immune microenvironment modulation by Ad5-ApoA1 were explored. ApoA1 expression was down-regulated with HCC progression and significantly positively correlated with the prognosis of HCC patients. Ad5-ApoA1 exhibited robust oncolytic activity but showed no therapeutic effect on nude mice. However, it significantly inhibited HCC growth and prolonged the survival period of both healthy-immune and humanized immune-reconstituted NCG mice. Furthermore, Ad5-ApoA1 significantly promoted the expression of IFN-γ and GzmB in CD8+ T cells while inhibiting the expression of PD-1 and LAG-3. Notably, the cholesterol content in the CD8+ T cells studied was significantly correlated with the expression of PD-1 and LAG-3, with ApoA1 promoting cholesterol efflux and reducing cholesterol levels. Ad5-ApoA1 activates CD8+ T cells by promoting large-scale viral replication. High levels of ApoA1 protein expression promote cholesterol efflux, inhibit CD8+ T cell depletion, and reduce inflammatory factors, ultimately leading to superior therapeutic effects on hepatocellular carcinoma.

Novel combination therapy using recombinant oncolytic adenovirus silk hydrogel and PD-L1 inhibitor for bladder cancer treatment

Recombinant oncolytic adenovirus offers a novel and promising cancer treatment approach, but its standalone efficacy remains limited. This study investigates a combination treatment strategy by co-administering recombinant oncolytic Adv-loaded silk hydrogel with a PD-L1 inhibitor for patients with bladder cancer to enhance treatment outcomes. Bladder cancer tissues from mice were collected and subjected to single-cell sequencing, identifying CRB3 as a key gene in malignant cells. Differential expression and functional enrichment analyses were performed, validating CRB3’s inhibitory role through in vitro experiments showing suppression of bladder cancer cell proliferation, migration, and invasion. Recombinant oncolytic adenoviruses encoding CRB3 and GM-CSF were constructed and encapsulated in silk hydrogel to enhance drug loading and release efficiency. In vivo experiments demonstrated that the nano-composite hydrogel significantly inhibited tumor growth and increased immune infiltration in tumor tissues. Co-administration of adenovirus silk hydrogel (Adv-CRB3@gel) with a PD-L1 inhibitor significantly enhanced T-cell infiltration and tumor killing. The combination of recombinant oncolytic Adv-loaded nano-composite hydrogel encoding CRB3 and GM-CSF with a PD-L1 inhibitor improves bladder cancer treatment outcomes by effectively recruiting T cells, providing a novel therapeutic strategy.

Oncolytic adenovirus encoding LHPP exerts potent antitumor effect in lung cancer

LHPP has been shown to be a new tumor suppressor, and has a tendency to be under-expressed in a variety of cancers. Oncolytic virotheray is a promising therapeutics for lung cancer in recent decade years. Here we successfully constructed a new recombinant oncolytic adenovirus GD55-LHPP and investigated the effect of GD55-LHPP on the growth of lung cancer cells in vitro and in vivo. The results showed that LHPP had lower expression in either lung cancer cells or clinical lung cancer tissues compared with normal cells or tissues, and GD55-LHPP effectively mediated LHPP expression in lung cancer cells. GD55-LHPP could effectively inhibit the proliferation of lung cancer cell lines and rarely affected normal cell growth. Mechanically, the oncolytic adenovirus GD55-LHPP was able to induce stronger apoptosis of lung cancer cells compared with GD55 through the activation of caspase signal pathway. Notably, GD55-LHPP also activated autophagy-related signal pathway. Further, GD55-LHPP efficiently inhibited tumor growth in lung cancer xenograft in mice and prolonged animal survival rate compared with the control GD55 or PBS. In conclusion, the novel construct GD55-LHPP provides a valuable strategy for lung cancer-targeted therapy and develop the role of tumor suppress gene LHPP in lung cancer gene therapy.

Apoptin Inhibits Glycolysis and Regulates Autophagy by Targeting Pyruvate Kinase M2 (PKM2) in Lung Cancer A549 Cells.

Pyruvate kinase M2 (PKM2) is a key enzyme in aerobic glycolysis and plays an important role in tumor energy metabolism and tumor growth. Ad-apoptin, a recombinant oncolytic adenovirus, can stably express apoptin in tumor cells and selectively causes cell death in tumor cells. The relationship between the anti-tumor function of apoptin, including apoptosis and autophagy activation, and the energy metabolism of tumor cells has not been clarified. In this study, we used the A549 lung cancer cell line to analyze the mechanism of PKM2 involvement in apoptin-mediated cell death in tumor cells. PKM2 expression in lung cancer cells was detected by Western blot and qRT-PCR. In the PKM2 knockdown and over-expression experiments, A549 lung cancer cells were treated with Ad-apoptin, and cell viability was determined by the CCK-8 assay and crystal violet staining. Glycolysis was investigated using glucose consumption and lactate production experiments. Moreover, the effects of Ad-apoptin on autophagy and apoptosis were analyzed by immunofluorescence using the Annexin v-mCherry staining and by western blot for c-PARP, p62, and LC3-II proteins. Immunoprecipitation analysis was used to investigate the interaction between apoptin and PKM2. In addition, following PKM2 knockdown and overexpression, the expression levels of p-AMPK, p-mTOR, p-ULK1, and p-4E-BP1 proteins in Ad-apoptin treated tumor cells were analyzed by western blot to investigate the mechanism of apoptin effect on the energy metabolism of tumor cells. The in vivo antitumor mechanism of apoptin was analyzed by xenograft tumor inhibition experiment in nude mice and immunohistochemistry of tumors' tissue. As a result, apoptin could target PKM2, inhibit glycolysis and cell proliferation in A549 cells, and promote autophagy and apoptosis in A549 cells by regulating the PKM2/AMPK/mTOR pathway. This study confirmed the necessary role of Ad-apoptin in the energy metabolism of A549 cells.

RCAd-LTH-shPD-L1, a double-gene recombinant oncolytic adenovirus with enhanced antitumor immunity, increases lymphocyte infiltration and reshapes the tumor microenvironment

BackgroundWith the successful development of modern immunotherapy, immune checkpoint inhibitors (ICIs) are currently considered potential therapeutic options for patients with cancer. However, the therapeutic potential of ICIs in human cancer...

A New HEK293 Cell with CR2 Region of E1A Gene Deletion Prevents the Emergence of Replication-Competent Adenovirus.

To eliminate the contaminants of Replication-Competent Adenovirus (RCA) during high titer recombinant oncolytic adenovirus production. At first, we detected E1A copy numbers of different sources of 293 cells using Q-PCR, and we screened a subclone JH293-C21 of the JH293 cell line (purchased from ATCC) with lower early region 1A (E1A) copy numbers and higher adenovirus production ability. Then, we deleted the conserved region (CR)2 of the E1A gene in this subclone using the CRISPR-Cas9 system and obtained a stable cell clone JH293-C21-C14 with lower E1A expression, but the RCA formation had no significant reduction. Then, we further deleted the CR2 of JH293-C21-C14 cells with the CRISPR-Cas9 system and obtained a strain of cells named JH293-C21-C14-C28. Finally, we detected the capacity for cell proliferation, adenovirus production, and RCA formation in the production of recombinant adenovirus. The JH293-C21-C14-C28 cells had a similar cell proliferation ability and human adenovirus production as JH293-C21 cells. Most importantly, RCA production in JH293-C21-C14-C28 cells was lower than in JH293-C21 cells. Human adenovirus producer cell clone JH293-C21-C14-C28 with CR2 deletion can effectively prevent the RCA production of replication-competent oncolytic adenovirus; this will provide significant advantages in utility and safety in gene therapy.

Recombinant oncolytic adenovirus armed with CCL5, IL-12, and IFN-γ promotes CAR-T infiltration and proliferation in vivo to eradicate local and distal tumors

The efficacy of chimeric antigen receptor T (CAR-T) cells for solid tumors remains unsatisfactory due to the limited tumor infiltration and immunosuppressive microenvironment. To overcome these limitations, the genetically engineered recombinant oncolytic adenoviruses (OAVs) that conditionally replicate in tumor cells were developed to modify the tumor microenvironment (TME) to facilitate CAR-T-mediated tumor eradication. Here in the present study, a novel recombinant OAV carrying CCL5, IL12, and IFN-γ controlled by Ki67 promoter was constructed (named AdKi67-C3). The antitumor activity of AdKi67-C3 was tested in vitro and in vivo by using mono administration or combing with CAR-T cells targeting B7H3. It proved that CCL5 expressed by AdKi67-C3 indeed induced more CAR-T migration in vitro and CAR-T infiltration in tumor mass in vivo. Meanwhile, cytokines of IFN-γ and IL12 secreted by AdKi67-C3-infected tumor cells significantly promoted proliferation and persistence of CAR-T cells in vitro and in vivo. In tumor-bearing xenograft mouse models of kidney, prostate or pancreatic cancer, local pretreatment with AdKi67-C3 dramatically enhanced CAR-T cell efficacy and eliminated local and distant tumors. More importantly, mice achieving complete tumor regression resisted to re-challenge with the same tumor cells, suggesting establishment of long-term antitumor immune response. Therefore, OAVs armored with cytokines could be developed as a bioenhancer to defeat the immunosuppressive microenvironment and improve therapeutic efficacy of CAR-T in solid tumors.

Hsa-microRNA-27b-3p inhibits hepatocellular carcinoma progression by inactivating transforming growth factor-activated kinase-binding protein 3/nuclear factor kappa B signalling

BackgroundMicroRNAs (miRNAs) play crucial roles in the development of hepatocellular carcinoma (HCC). Hsa-microRNA-27b-3p (hsa-miR-27b) is involved in the formation and progression of various cancers, but its role and clinical value in HCC remain unclear.MethodsThe expression of hsa-miR-27b in HCC was examined by quantitative real-time PCR (qRT-PCR) and in situ hybridization (ISH) assays of clinical samples. Cell Counting Kit-8 assays (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU) incorporation assays, Transwell assays, filamentous actin (F-actin) staining and western blot analyses were used to determine the effects of hsa-miR-27b on HCC cells in vitro. Subcutaneous xenograft and lung metastatic animal experiments were conducted to verify the role of hsa-miR-27b in HCC in vivo. In silico prediction, qRT-PCR, western blot, anti-Argonaute 2 (AGO2) RNA immunoprecipitation (RIP) and dual luciferase reporter assays were applied to identify the target genes of hsa-miR-27b. To detect the impacts of hsa-miR-27b on nuclear factor kappa B (NF-кB) signalling cascades mediated by transforming growth factor-activated kinase-binding protein 3 (TAB3), we performed qRT-PCR, western blot assays, immunofluorescence staining, immunohistochemistry (IHC) and dual-luciferase reporter assays. Recombinant oncolytic adenovirus (OncoAd) overexpressing hsa-miR-27b was constructed to detect their therapeutic value in HCC.ResultsThe expression of hsa-miR-27b was lower in HCC than in adjacent non-tumourous tissues (ANTs), and the reduced expression of hsa-miR-27b was associated with worse outcomes in patients with HCC. Hsa-miR-27b significantly inhibited the proliferation, migration, invasion, subcutaneous tumour growth and lung metastasis of HCC cells. The suppression of hsa-miR-27b promoted the nuclear translocation of NF-κB by upregulating TAB3 expression. TAB3 was highly expressed in HCC compared with ANTs and was negatively correlated with the expression of hsa-miR-27b. The impaired cell proliferation, migration and invasion by hsa-miR-27b overexpression were recovered by ectopic expression of TAB3. Recombinant OncoAd with overexpression of hsa-miR-27b induced anti-tumour activity compared with that induced by negative control (NC) OncoAd in vivo and in vitro.ConclusionsBy targeting TAB3, hsa-miR-27b acted as a tumour suppressor by inactivating the NF-кB pathway in HCC in vitro and in vivo, indicating its therapeutic value against HCC.Graphical

A comparative study of the ability of recombinant oncolytic adenovirus, doxorubicin and tamoxifen to inhibit the proliferation of breast cancer cells.

In this study, we compared the inhibitory effects of recombinant oncolytic adenovirus (Ad‐apoptin‐hTERTp‐E1a, Ad‐VT) with that of doxorubicin (DOX), a first‐line chemotherapy drug, and tamoxifen (TAM), an endocrine therapy drug, on the proliferation of breast cancer cells. We found that Ad‐VT could effectively inhibit the proliferation of breast cancer cells (p < 0.01); the inhibition rate of Ad‐VT on normal mammary epithelial MCF‐10A cells was less than 20%. DOX can effectively inhibit the proliferation of breast cancer cells and also has a strong inhibitory effect on MCF‐10A cells (p < 0.01). TAM also has a strong inhibitory effect on breast cancer cells, among which the oestrogen‐dependent MCF‐7 cell inhibition was stronger (p < 0.01), At higher concentrations, TAM also had a high rate of inhibition (>70%) on the proliferation of MCF‐10A cells. We also found that both recombinant adenovirus and both drugs could successfully induce tumour cell apoptosis. Further Western blot results showed that the recombinant adenovirus killed breast cancer cells through the endogenous apoptotic pathway. Analysis of the nude mouse subcutaneous breast cancer model showed that Ad‐VT significantly inhibited tumour growth (the luminescence rate of cancer cells was reduced by more than 90%) and improved the survival rate of tumour‐bearing mice (p < 0.01). Compared with DOX and TAM, Ad‐VT has a significant inhibitory effect on breast cancer cells, but almost no inhibitory effect on normal breast epithelial cells, and this inhibitory effect is mainly through the endogenous apoptotic pathway. These results indicate that Ad‐VT has significant potential as a drug for the treatment of breast cancer.

Recombinant Oncolytic Adenovirus Combined with Cyclophosphamide Induces Synergy in the Treatment of Breast Cancer in vitro and in vivo.

PurposeOncolytic virus therapy has gradually become an integral approach in cancer treatment. We explored the therapeutic effects of the combination of a dual cancer-selective anti-tumor recombinant adenovirus (Ad-Apoptin-hTERTp-E1a) and cyclophosphamide on breast cancer cells.MethodsThe inhibition of MCF-7 and MDA-MB-231 breast cancer cells by Ad-Apoptin-hTERTp-E1a (Ad-VT), cyclophosphamide, and Ad-VT + Cyclophosphamide was investigated using the CCK-8 assay. The combination index (CI) was calculated using CalcuSyn software to determine the best combination based on the inhibition rates of the different treatment combinations. The CCK-8 assay and crystal violet staining were used to detect the cytotoxicity of the combined Ad-VT and cyclophosphamide in breast cancer cells and breast epithelial cells. Subsequently, Hoechst staining, annexin V flow cytometry, and JC-1 staining were used to analyze the inhibitory pathway of Ad-VT plus cyclophosphamide on breast cancer cells. Cell migration and invasion of breast cancer cells were assessed using the cell-scratch and Transwell assays. The anti-tumor effects of different treatment groups in a tumor-bearing nude mouse model also were analyzed.ResultsThe treatment combination of Ad-VT (40 MOI) and cyclophosphamide (400 µM) significantly inhibited MCF-7 and MDA-MB-231 cells and reduced the toxicity of cyclophosphamide in normal cells. Ad-VT primarily induced breast cancer cell apoptosis through the endogenous apoptotic pathway. Apoptosis was significantly increased after treatment with Ad-VT plus cyclophosphamide. The combination significantly inhibited the migration and invasion of MCF-7 and MDA-MB-231 cells. The in vivo experiments demonstrated that exposure to Ad-VT plus cyclophosphamide significantly inhibited tumor growth and extended the survival time of the nude mice.ConclusionAd-VT plus cyclophosphamide reduced toxicity and exhibited increased efficacy in treating breast cancer cells.

CXCL10-armed oncolytic adenovirus promotes tumor-infiltrating T-cell chemotaxis to enhance anti-PD-1 therapy

ABSTRACT Resistance remains an obstacle to anti-programmed cell death protein 1 (PD-1) therapy in human cancer. One critical resistance mechanism is the lack of T cell chemotaxis in the tumor microenvironment (TME). CXCL10-CXCR3 signaling is required for T cell tumor infiltration and tumor immunotherapy. Oncolytic viruses (OVs), including oncolytic adenoviruses (AdVs), induce effective T cell immunity and tumor infiltration. Thus, arming OV with CXCL10 would be an attractive strategy to overcome resistance to anti-PD1 therapy. Here, we successfully constructed a novel recombinant oncolytic adenovirus encoding murine CXCL10, named Adv-CXCL10. Through intratumoural injection, the continuous expression of the functional chemokine CXCL10 in the TME is realized to recruit more CXCR3+ T cells into the TME to kill tumor cells, and the recombinant adenovirus shows great power to ‘fire up’ the TME and enhance the antitumour efficiency of PD-1 antibodies.

Combined oncolytic adenovirus carrying MnSOD and mK5 genes both regulated by survivin promoter has a synergistic inhibitory effect on gastric cancer.

Gastric cancer (GC) is one of the major causes of cancer-related mortality. The use of oncolytic virus for cancer gene-virotherapy is a new approach for the treatment of human cancers. In this study, a novel Survivin promoter-driven recombinant oncolytic adenovirus carrying mK5 or MnSOD gene was constructed, which was modified after deletion of the E1B gene. Human plasminogen Kringle 5 mutant (mK5) and manganese superoxide dismutase (MnSOD) are both potential tumor suppressor genes. By constructing Ad-Surp-mK5 and Ad-Surp-MnSOD oncolytic adenoviruses, we hypothesized that the combination of the two viruses would enhance the therapeutic efficacy of GC as compared to the one virus alone. The results of the in vitro experiments revealed that the combination of adenovirus carrying mK5 and MnSOD gene exhibited stronger cytotoxicity to GC cell lines as compared to the virus alone. Additionally, the virus could selectively kill cancer cells and human somatic cells. Cell staining, flow cytometry, and western blot analysis showed that the combination of two adenoviruses containing therapeutic genes could promote the apoptosis of cancer cells. In vivo experiments further verified that Ad-Surp-mK5 in combination with Ad-Surp-MnSOD exhibited a significant inhibitory effect on the growth of GC tumor xenograft as compared to the virus alone, and no significant difference was observed in the bodyweight of treatment and the normal mice. In conclusion, the combination of our two newly constructed recombinant oncolytic adenoviruses containing mK5 or MnSOD therapeutic genes could significantly inhibit gastric cancer growth by inducing apoptosis, suggestive of its potential for GC therapy.

Ad-apoptin inhibits glycolysis, migration and invasion in lung cancer cells targeting AMPK/mTOR signaling pathway

Ad-apoptin is a recombinant oncolytic adenovirus constructed by our laboratory that can express apoptin. It can selectively kill tumor cells without damaging normal cells. This study investigated the effects of Ad-apoptin on glycolysis, migration and invasion of non-small cell lung cancer. Cell viability and apoptosis were detected by CCK-8 and flow cytometry, respectively. Glycolysis was investigated by glucose consumption, lactic acid production and glycolytic key enzyme protein levels. Migration and invasion were evaluated via wound healing, transwell assays and epithelial-mesenchymal transition (EMT) protein levels. The interaction between apoptin and AMPK was detected by Co-IP. A nude mice tumor model was established to investigate the anti-cancer role of Ad-apoptin in vivo. The results showed that Ad-apoptin inhibits cell viability and induces apoptosis of A549 and NCI–H23 cells. Ad-apoptin can reduce the glucose uptake and lactic production in lung cancer cells, and reduce the expression of related glycolysis-limiting enzymes. At the same time, Ad-apoptin inhibited the migration and invasion of lung cancer. Immunoprecipitation showed that apoptin and AMPK could interact directly. Moreover, knockdown of AMPK significantly attenuated the inhibitory effect of Ad-apoptin on glycolysis, migration and invasion of A549 and NCI–H23 cells. Ad-apoptin can inhibit the growth of tumors in nude mice. Compared with the control group, Ad-apoptin had a significant inhibitory effect on AMPK knockdown tumors. The immunohistochemical results of tumor tissues were consistent with those in vitro. Collectively, Ad-apoptin targets AMPK and inhibits glycolysis, migration and invasion of lung cancer cells through the AMPK/mTOR signaling pathway. This suggests that Ad-apoptin may have therapeutic potential for lung cancer by targeting AMPK activation.

An Oncolytic Adenovirus Armed with a Nanobody Against CD47 Reprograms Tumor Immune Microenvironment and Drives Durable Antitumor Immunity

Background: Oncolytic virotherapy utilizes natural or engineered viruses to kill tumors selectively, representing a promising cancer immunotherapy. In addition to direct oncolysis, oncolytic viruses (OVs) elicit potent and durable antitumor immune responses by induction of immunogenic cell death of tumors. Membrane protein CD47 overexpressed on tumor cells engages in don't eat me signal that prevents macrophages from engulfing tumor cells. CD47-targeting agents have been tested via preclinical and clinical trials. As ideal gene delivery vectors in cancer therapy, OVs can be engineered to express anti-CD47 antibodies to induce potentiated tumor killing. Importantly, OVs can intratumorally deliver anti-CD47 antibodies without causing uncontrolled off-tumor toxicity. Methods: We developed a recombinant oncolytic adenovirus loaded with a CD47-targeting nanobody fused with the IgG2a Fc protein. An oncolytic adenovirus without transgene of interest was used as control. B16-F10 melanoma, A20 lymphoma, and 4T1 breast cancer mouse models were established to evaluated in vivo antitumor efficacy of oncolytic adenoviruses. Findings: The recombinant oncolytic adenovirus armed with a nanobody against CD47 induced durable suppression of the tumor and long-term survival of tumor-bearing mice. Additionally, the recombinant oncolytic adenovirus elevated the number of tumor-infiltrating immune cells with an activated immunophenotype, suggesting that it could remodel the tumor immune microenvironment. Systemic antitumor effects and immune memory were also observed in mice treated with the anti-CD47 oncolytic adenovirus; tumorigenesis was completely inhibited in these mice after tumor re-challenge. Interpretation: The recombinant anti-CD47 oncolytic adenovirus has an effectual antitumor activity and may be a promising antitumor agent. Funding Statement: This work was supported by the National Science and Technology Major Projects of New Drugs (2018ZX09201018-013), the National Science and Technology Major Project for Infectious Diseases Control (2017ZX10203206-004), the National Natural Science Foundation of China (81101728), Sichuan Regional Innovation Cooperation Project (20QYCX0100), the Innovation Spark Project of Sichuan University (2018SCUH0084). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: All animal experiments were approved by the Institutional Animal Care and Use Committee of Sichuan University, Chengdu, China.

Cytokine-induced killer cells carrying recombinant oncolytic adenovirus expressing p21Ras scFv inhibited liver cancer

Background: Oncolytic adenovirus-mediated gene therapy is an emerging strategy for cancer treatment. However, oncolytic adenoviruses are mainly administered locally at tumor site. Intravenous administration of oncolytic adenovirus for cancer gene therapy is a problem that needs to be solved urgently.Methods: We constructed recombinant oncolytic adenovirus KGHV500 carrying anti-p21Ras scFv and employed CIK cells to deliver KGHV500. TUNEL, wound healing, MTT, and Transwell invasion assays were used to determine the anti-tumor efficacy of KGHV500 on liver cancer cells in vitro. Nude mouse xenograft model was used to examine the anti-tumor efficacy of CIK cells combined with KGHV500 in vivo. Furthermore, KGHV500 accumulation in different organs was detected to assess the safety.Results: KGHV500 inhibited the migration, proliferation, invasion, and induced the apoptosis of liver cancer cells. CIK cells carrying KGHV500 reached tumor site and exerted much better anti-tumor efficacy than CIK cells or KGHV500 alone in nude mouse xenograft model. Moreover, we detected KGHV500 and anti-p21Ras scFv in different organs of nude mice, with little effects on the organs.Conclusions: We develop a novel strategy for the treatment of Ras-driven liver cancer by combining CIK cells with oncolytic adenovirus expressing anti-p21Ras scFv. Intravenous injection of CIK cells carrying KGHV500 in vivo significantly inhibits tumor growth, has little effect on normal organs, and is relatively safe.

Antitumor effect of hypoxia response element chimeric tumor specific promoter regulated oncolytic adenovirus on renal cell carcinoma

Objective To investigate the effect of chimeric promoter composed of hypoxia response element (HRE) and tumor specific cell proliferation-associated nuclear antigen (Ki-67) promoter on the replication ability of oncolytic adenovirus and its cytotoxic activity under hypoxic conditions. Methods The HRE sequence, as an enhancer, was inserted upstream of the Ki-67 promoter and, at the same time carried the reporter gene enhanced green fluorescent protein (EGFP) to construct oncolytic adenovirus Ad-HRE-Ki-67-EGFP, and the titer of adenoviruses was measured by the tissue culture infective dose (TCID50). Normal renal epithelial cells HK-2 and renal cancer cells OSRC-2 and ACHN were infected with two kinds of viruses under normoxic (21% O2) or hypoxic (1% O2) conditions respectively for 24 h, and the virus tumor-targeting was detected by fluorescence microscopy and flow cytometry. Western blotting was performed to detect the expression of hypoxia-related protein hypoxia-inducible factor-1 alpha (HIF-1α), vascular endothelial growth factor (VEGF) and the expression of E1A protein. Cell counting kit-8 (CCK-8) assay was used to detect the cytotoxity of virus on cells. Results The recombinant oncolytic virus Ad-HRE-Ki67-EGFP was successfully constructed. Under normoxic conditions, the efficiency of ACHN and OSRC-2 infection by chimeric HRE oncolytic adenovirus in renal cancer cells was (47.5±2.6)% and (35.2±3.4)%, while that in hypoxic conditions was (86.4±3.4)% and (73.5±2.3)%, indicating that the infection efficiency was higher under hypoxic conditions (ACHN: t=15.760; OSRC-2: t=16.190; P<0.01); Normal renal tubular epithelial cells HK-2 were infected with Ad-Ki-67-EGFP or Ad-HRE-Ki-67-EGFP under normoxic conditions. The infection efficiency was (22.4±3.1)% and (22.9±2.2)% respectively, compared with that of ACHN and OSRC-2 cells [ACHN: (68.4±3.3)% and (72.1±2.9)%; OSRC-2: (56.1±3.1)% and (61.4±2.3)%], the difference was statistically significant (Ad-Ki-67-EGFP: t=17.650, 13.390; Ad-HRE-Ki-67-EGFP: t=23.680, 21.430, P<0.01), indicating that the two adenoviruses have a targeting effect on renal cancer cells. Western blotting data showed that the expression of HIF-1α, VEGF and p53 protein were increased under hypoxic conditions and was time-dependent, indicating that hypoxia can regulate the transcription and translation of HIF-1α, VEGF and p53 proteins. Under hypoxic conditions, the expression level of E1A protein in OSRC-2 and ACHN infected with Ad-HRE-Ki67-EGFP was significantly higher than that after Ad-Ki67-EGFP infection. It indicated that HRE can enhance the replication ability of virus under hypoxic conditions. CCK-8 results showed that when the multiple infection (MOI) values were 20, 100, the survival rate of the Ad-HRE-Ki-67-EGFP-normoxia virus group in ACHN and OSRC-2 cells was (73.4±2.0)%, (56.4±1.5)% and (79.9±1.8)%, (61.3±2.7)%, the Ad-HRE-Ki-67-EGFP-hypoxia virus group was (63.1±2.0)%, (31.6±2.1)% and (68.1±2.6)%, (35.8±3.1)%, the Ad-HRE-Ki-67-EGFP-hypoxia virus group showed a stronger proliferation inhibitory effect [MOI (20): t=6.328, 6.441; MOI (100): t=16.410, 10.790, P<0.01]. Conclusion Under hypoxic conditions, chimeric HRE sequence recombinant oncolytic adenovirus has stronger replication ability and cell killing effect in renal carcinoma cells. Key words: Renal cell carcinoma; Oncolytic adenovirus; Hypoxia response element; Hypoxia

Recombinant Adenovirus KGHV500 and CIK Cells Codeliver Anti-p21-Ras scFv for the Treatment of Gastric Cancer with Wild-Type Ras Overexpression

The development of gastric cancer is frequently related to the overexpression of wild-type p21 proteins, but it is rarely related to mutated Ras proteins. We previously constructed a broad-spectrum anti-p21-Ras single-chain variable fragment antibody (scFv), which was carried by the oncolytic adenovirus KGHV500. Here we explored the antitumor effects of this recombinant oncolytic adenovirus carried by cytokine-induced killer (CIK) cells on human gastric SGC7901 cells that overexpress wild-type Ras. The MTT assay, scratch test, Transwell assay, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were performed in vitro to investigate the proliferation, migration, invasiveness, and cell apoptosis rate, respectively, of the human gastric cell line SGC7901 treated with KGHV500 adenovirus. Then, the tumor-targeting ability and systemic safety of KGHV500 adenovirus delivered by CIK cells were explored in vivo. We found that KGHV500 adenovirus could significantly inhibit proliferation, migration, and invasiveness and promote cell apoptosis in SGC7901 cells in vitro. In vivo studies showed that CIK cells could successfully deliver KGHV500 adenovirus to the tumor site; the two vectors synergistically killed tumor cells, and the treatment was relatively safe for normal tissues. In conclusion, this therapeutic strategy of recombinant adenovirus KGHV500 delivered by CIK cells offers a positive prospect for the targeted therapy of Ras-related cancers.

Effects and mechanism of recombinant oncolytic adenovirus expressing soluble programmed cell death protein 1 on proliferation and apoptosis of B16/F10 cell

Objective To construct a recombinant oncolytic adenovirus expressing soluble programmed cell death protein 1 (sPD1) and investigate its effects and mechanism on proliferation and inhibition of B16/F10 cells. Methods The recombinant oncolytic adenovirus Ad5-sPD1 was constructed, and infected into B16/f10 cells. The quantitative fluorescent polymerase chain reaction was used to detect the viral replication. MTT assay was applied to examine the inhibition of B16/F10 cells. The viruses were injected subcutaneously into the mouse B16/F10 tumor model to study the antitumor effect of Ad5-sPD1 in vivo. EliSpot technique was exployed to test the number of tumor proliferative lymph nodes. Results After viral treatment of B16/F10 cells for 6, 24, 36, 48, 60 and 72 h, the copies fold increases in the Ad5 treatment group were respectively 0.99±0.10, 1.12±0.05, 1.57±0.15, 3.59±1.03, 6.26±0.74, 9.69±1.46, and those in the Ad5-sPD1 treatment group were respectively 1.00±0.104, 0.99±0.08, 1.05±0.03, 3.56±1.04, 6.04±0.38, 11.05±1.12. There was no significant difference between the two groups (P=0.754). After treating B16/F10 cells for 72 h with multiplicity of infection (MOI) of 0, 5, 10, 15 and 20, the cell viability of Ad5 treatment group was respectively (100.00±6.39)%, (101.60±5.21)%, (98.66±7.31)%, (93.50±4.54)%, (73.16±2.45)%, and that in the Ad5-sPD1 treatment group was respectively (100.00±6.38)%, (97.10±6.37)%, (96.96±7.44)%, (95.67±7.46)%, (74.49±4.14)%. There was no significant difference between the two groups (P=0.779). After treating mice model of B16/F10 subcutaneous tumor with Ad5 and Ad5-sPD1 viruses, tumor volume was respectively 3 638±1.823, 2 603±336 and 1 097±532 at the termination of the experiments with the difference being significant (Mock and Ad5: P=0.044; Mock and Ad5-sPD1: P=0.000; Ad5 and Ad5-sPD1: P=0.000). After respectively injecting mice model of B16/F10 subcutaneous tumor with Mock, Ad5 and Ad5 -sPD1 viruses, the number of IFN-γ spots was respectively 19.4±7.75, 149.8±25.18 and 314±32.34 with the the difference being significant (Mock and Ad5: P=0.000; Mock and Ad5-sPD1: P=0.000; Ad5 and Ad5-sPD1: P=0.000). Conclusion Recombinant oncolytic adenovirus expressing PD1 gene can significantly improve the anti-tumor effect of the oncolytic adenovirus by blocking PD-1/PD-L1 inhibitory pathway. Key words: Oncolytic adenovirus; Tumor; Immune response; Programmed cell death protein 1

Stability analysis on the radioactive iodine-labelled prostate cancer-specific recombinant oncolytic adenovirus.

The aim of the present study was to construct the 125I-replication-selective oncolytic adenovirus (RSOAds)-human telomerase reverse transcriptase (hTERT)/prostate specific antigen (PSA) nuclide-oncolytic virus marker by labelling the hTERT/PSA double-regulation replicative oncolytic adenovirus with 125I nuclide, and investigate the influence of viral markers under various reaction conditions on labelling efficiency. N-bromosuccinimide (NBS) was used as the oxidizer for 125I labelling, and the best conditions for labelling were identified through the reactions between oncolytic adenovirus at various concentrations and NBS. Dosage of 125I, reaction duration, pH values and reaction volume were respectively evaluated to determine their effects on the labelling efficiency of 125I-RSOAds-hTERT/PSA nuclide-oncolytic adenovirus markers. Purified nuclide-oncolytic adenovirus markers were isolated by gel-filtration chromatography; paper chromatography was performed to assay the radiochemical purity of 125I-RSOAds-hTERT/PSA markers at various time points. Radiochemical purity of 125I-RSOAds-hTERT/PSA was >95%, and could be maintained at 4°C for 7 days. The best reaction conditions were set as follows: 0.5 µl of 125I (~0.2 m Ci, 7.4 MBq); 25 qg of NBS; 100 µl of 8×109 VP/ml 125I-RSOAds-hTERT/PSA virus solution; 30 min of reaction duration; pH 7.5; 120 µl of PBS. Labelling hTERT/PSA double-regulation replicative oncolytic adenovirus with 125I was identified to be available, and the radiochemical purity of acquired virus markers could be maintained under specific conditions.