By introducing the concept of genetically encoded BRET - activated PDT , we propose a viable solution of the bottleneck problem of limited treatment depth of PDT , while avoiding problems of currently available chemically assembled BRET complexes . For the demonstration of the concept , we designed a genetically encoded NanoLuc - miniSOG platform , which can be delivered by a properly selected carrier to a tumor , and then triggered by the injection of furimazine substrate . To assess the efficiency of this pair , we expressed NanoLuc - miniSOG BRET - pair in breast ductal carcinoma BT-474 cancer cells and then examined its operation in vitro and in vivo . Spectroscopic characterization of BT-474 / NanoLuc - miniSOG cell line confirmed that BRET between NanoLuc and miniSOG    c Tumor - growth curves for control group ( mice treated with PBS only ) , as well as for groups carrying NanoLuc or NanoLuc - miniSOG LVs particles and treated with furimazine . Data are presented as the mean ± SD ( n = 6 ) . d In vivo bioimaging of animals bearing HER2 - positive tumors and injected i.t . with LVs - NanoLuc - miniSOG or LVs - NanoLuc . Three mice from each group on the 13th day after LVs injection are presented indeed occurs with the BRET ratio reaching 0.74 ± 0.05 ( Fig . 1d ) . The recorded ratio is in agreement with our previous data 23 , as well as with data for other BRET - based genetically encoded sensor systems 33,34 , confirming a high efficiency of the pair . We demonstrated by MTT assays that NanoLuc - miniSOG efficiently kills the cancer cells in the presence of furimazine ( Fig . 1e ) , while other tests confirmed that the endogenous bioluminescence can serve as a light source to activate the miniSOG for ROS generation ( Fig . 1f ) . Using the model of mice bearing engineered BT-474 cells expressing NanoLuc - miniSOG , we recorded TGI exceeded 72 % after the injection of furimazine without external light irradiation ( Fig . 2 ) . It means that the inhibition of tumor growth occurs due to BRET - induced PDT , although cytotoxicity of oxidized product of furimazine could also partially contribute to the cell death ( Fig . 2b ) . It is also important that bioluminescence imaging can be used in our case to assess BRET efficiency in vivo . The differences of the average luminescence signal with or without Rf in animals evidenced an efficient process of energy transfer from the donor ( oxidized NanoLuc luciferase - substrate ) to the acceptor ( miniSOG ) in the case of tumor Rf saturation ( Fig . 2c , d ) . Finally , genetic nature of proposed NanoLuc - miniSOG BRET - pair allowed us to use targeted viral system for gene delivery precisely into tumor ( Fig . 4 ) . Using HER2 - specific LVs carrying NanoLuc - miniSOG gene we achieved significant regression ( TGI 67 % ) of HER2 - positive xerograph tumor in the animal model , confirming a high efficiency of the proposed concept . We foresee several major advantages of the proposed concept of genetically encoded BRET - activated PDT over currently present chemically assembled BRET - based systems for PDT . First , the genetic nature of functional PDT elements provides an ideal spatial architecture of the BRET construct , conditioning optimal distance between the internal triggering source and the PS , which is obviously very difficult or impossible in the case of chemical assembling . Second , the proposed approach does not require any sophisticated and costly chemical conjugation protocols to generate functional BRET pair for PDT . Third , as showed in our studies , despite certain local toxic effects in organs due to furimazine injection , genetic encoding nature of synthesized protein complexes makes them safe and easily excretable from the organism , which is not the case for many chemically assembled structures . Finally , as probably the main advantage , the used principle of genetic encoding opens up novel appealing prospective for future improvements of PDT systems profiting from on genetic engineering approaches . In particular , the expression of used NanoLuc - miniSOG pair can be controlled on the genetic level by properly designed promoters ( e.g. , telomerase promoters ) , which are specific to some tumors . In addition , by fusing with a well - known protein localization motif ( nuclear , membrane , mitochondrial ) or a whole protein , the genetically encoded pair can be easily directed to any cell compartment ( or even sub - compartment ) . Furthermore , the use of different targeting molecules in the composition of lentiviruses or other carriers renders possible an easy re - direction of a genetically encoded BRET - activated system on any tumor type , while the therapeutic action will address not only the primary tumor , but also its metastasis distributed in the organism . Thus , the employment of genetically encoded constructs provides novel opportunities to direct BRETinduced generation of active forms of oxygen to different cellular compartments or particular cell lines , which is hardly possible with chemically assembled BRET systems .