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Oncolytic Adenovirus Programmed by Synthetic Gene Circuit for Cancer Immunotherapy

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Oncolytic Adenovirus Programmed by Synthetic Gene Circuit for Cancer Immunotherapy ARTICLE https://doi.org/10.1038/s41467-019-12794-2 OPEN Oncolytic adenovirus programmed by synthetic gene circuit for cancer immunotherapy Huiya Huang1,4, Yiqi Liu2,4, Weixi Liao1,4, Yubing Cao2, Qiang Liu2, Yakun Guo2, Yinying Lu3 & Zhen Xie1* Improving efficacy of oncolytic virotherapy remains challenging due to difficulty increasing specificity and immune responses against cancer and limited understanding of its population dynamics. Here, we construct programmable and modular synthetic gene circuits to control 1234567890():,; adenoviral replication and release of immune effectors selectively in hepatocellular carcinoma cells in response to multiple promoter and microRNA inputs. By performing mouse model experiments and computational simulations, we find that replicable adenovirus has a superior tumor-killing efficacy than non-replicable adenovirus. We observe a synergistic effect on promoting local lymphocyte cytotoxicity and systematic vaccination in immunocompetent mouse models by combining tumor lysis and secretion of immunomodulators. Furthermore, our computational simulations show that oncolytic virus which encodes immunomodulators can exert a more robust therapeutic efficacy than combinatorial treatment with oncolytic virus and immune effector. Our results provide an effective strategy to engineer oncolytic adenovirus, which may lead to innovative immunotherapies for a variety of cancers. 1 MOE Key Laboratory of Bioinformatics and Bioinformatics Division, Center for Synthetic and System Biology, Department of Automation, Beijing National Research Center for Information Science and Technology, Tsinghua University, Beijing 100084, China. 2 Syngentech Inc., Zhongguancun Life Science Park, Changping District, Beijing 102206, China. 3 The comprehensive Liver cancer center, The 5th medical center of PLA Genaral Hospital, 100 Xi-Si-Huan Middle Road, Beijing 100039, China. 4These authors contributed equally: Huiya Huang, Yiqi Liu, Weixi Liao. *email: [email protected] NATURE COMMUNICATIONS | (2019) 10:4801 | https://doi.org/10.1038/s41467-019-12794-2 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-12794-2 ecent clinical success has demonstrated the great potential composed of two mutually inhibiting transcription activator-like of cancer immunotherapy to overcome tumor-mediated effector repressors (TALERs) can function as a robust switch R 26,27 immunosuppression. However, the development of controlled by microRNA inputs . To further improve the immunotherapeutic antibodies and chimeric antigen receptor cancer-targeting specificity of OV and trigger immune response (CAR)-T cells are limited by the lack of targetable tumor against cancer cells in the cancer microenvironment, we engineer antigens1,2. In addition, therapeutic efficacy of individual a compact (<8.5-kb) and programmable switch circuit to control immunomodulators such as cytokines, chemokines and check- the replication of OV and expression of immune effectors point inhibitors is often compromised when infiltrated lympho- (Fig. 1a). In this setup, a transcription activator driven by a cytes are limited in tumor microenvironment3. Systemic cancer-specific promoter turns on two mutually inhibiting treatment of immunomodulators either alone or in combinations repressors that are respectively repressed by two microRNAs frequently cause severe side effect4. These studies highlight a (miR-a and miR-b). The switch-controlled E1A and immune continuous advancement and a large unmet demand in cancer effector genes co-express by using a self-cleavage 2A linker when immunotherapy to explore efficient approaches to locally mod- the activator and miRNA-b are at high levels and miRNA-a is at ulate immunological state in tumor microenvironment with low level (Fig. 1b)28. reduced side effect. To realize the application potential of synthetic gene circuits Oncolytic virus is an attenuated or engineered virus that can for identification and destroy of cancer cells, it is essential to cause cancer cell lysis due to selective replication, and trigger develop an appropriate delivery vector platform. In this study, we systematic immune responses, which has been considered as a develop a sensory switch circuit which is about 6.5 kb in length class of medicines for cancer immunotherapy5. Although many and establish a hierarchical assembly method to efficiently load oncolytic viruses alone only cause weak immune responses, the circuits into adenoviral vector backbone, which can control therapeutic efficacy of oncolytic virus against tumors can be adenoviral replication and coexpression of immune effectors, greatly enhanced by encoding and locally releasing cytokines and such as human GM-CSF, interleukin-2 (IL-2), single-chain vari- chemokines, which helps overcoming immunosuppression in able fragments (scFvs) against either programmed death-1 (PD-1) tumor microenvironment with reduced side effect compared to or programmed death-ligand 1 (PD-L1). It has been shown that systematic administration of immunomodulators6–9. In addition, viral-encoded immune effectors can promote anti-cancer cyto- recent study shows that oncolytic virus (Talimogene laherpar- toxicity of oncolytic virotherapy by modulating tumor micro- epvec) that encodes human granulocyte-macrophage colony-sti- environment29–31. For proof-of-concept, we aim to construct the mulating factor (GM-CSF) can dramatically increase the objective programmable sensory switch to target hepatocellular carcinoma response rate of programmed death protein 1 (PD-1) inhibitor (HCC) cells because HCC is the major type of liver cancers with therapy (pembrolizumab) independent on the baseline CD8+ large unmet clinical needs. We demonstrate the modularity of our lymphocyte infiltration, highlighting the potential of oncolytic circuit design by changing immunomodulatory genes, and con- virus to change and reactivate the local immune microenviron- firm the high selectivity of programmed oncolytic virus to kill a ment against tumors10. However, it is still a great challenge to variety of HCC cancer cells in cell cultures and in mouse models. increase tumor-targeting specificity and immuno-stimulation Unlike traditional drugs with defined pharmacokinetics and efficacy of oncolytic virus. pharmacodynamics, the outcome of oncolytic virus immu- The cancer-targeting specificity of oncolytic virus is also critical notherapy relies on the population dynamics and interactions of for the safety and efficacy of oncolytic virus immunotherapy. A tumor cells, viruses and surrounding immune cells, which makes variety of viruses have been engineered as oncolytic viruses, such it challenging to systematically understand important features of as adenovirus, herpes simplex virus (HSV), and vaccinia oncolytic virotherapy. Mathematical models can be helpful to virus6,11,12. Adenovirus that contains a double-stranded linear analyze dynamics of complex therapeutic system32. Inspired by DNA genome of ~36 kb, becomes attractive due to low patho- previous studies33,34, we establish coarse-grained models to genic risk, high genome stability, wide range of tissue tropism quantitatively describe the system of oncolytic virotherapy in and relatively large DNA loading capacity (up to 8.5-kb foreign three different scenarios, where the lymphocytes are absent, or DNA)5,7,13. In the adenoviral genome, the E1 region encodes E1A present with or without immunomodulators. and E1B proteins, and induction of E1A expression alone may be In all, this study assembles programmed OVs with sensory sufficient to initiate the virus replication in different cancer gene circuit switch that exert high selectivity to kill HCC in cell cells14. The selectivity of oncolytic adenovirus (OV) can be cultures and mouse models. Through mathematical model, this improved by simply using cancer-specific promoters or micro- study identifies key characteristics of oncolytic virotherapy and RNAs to regulate the E1A expression7,8,15,16. However, it is still provides guideline for future improvements. necessary to further reduce the pathogenicity of adenovirus to ensure the safety in clinical applications. An appealing strategy is to combine multiple inputs to further improve the cancer- Results targeting specificity17. In addition, the loading capacity of Characterization of sensory switch circuits. Because the loading replication-competent adenoviral vector limits accommodation of capacity of replication-competent adenoviral vector is up to sophisticated gene circuits and modifying OV is still costly and ~8.5 kb, we constructed sensory switch circuit by using gene inefficient by using a ‘trial-and-error’ method, which demands an parts with a reduced size. Consistence with previous reports35, effective engineering framework to facilitate the development of by using quantitative RT-PCR we confirmed that α-fetoprotein OV18,19. (AFP) gene expressed in several types of HCC cells but not in Built on engineering principles, an important aim of mam- Chang cells originally derived from normal human liver tissue malian synthetic biology is to construct synthetic gene circuits (Supplementary Fig. 1a). To reduce the length of AFP pro- with modular and standardized parts for sophisticated informa- moter, we constructed 5 truncated AFP promoter derivatives tion processing and programmable regulations inside live cells. and analyzed the promoter activity by using transient trans- Several mammalian gene circuits have been engineered to inte- fection. We found that the EYFP expression driven
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