ABSTRACT
Prodrugs are harmless until activated by a bacterial or viral gene product; they constitute the basis of gene-delivered prodrug therapies called GDEPT, which can kill tumors without major side effects. Previously, we utilized the prodrug CNOB (C16H7CIN2O4; not clinically tested) and enzyme HChrR6 in GDEPT to generate the drug MCHB (C16H9CIN2O2) in tumors. Extracellular vesicles (EVs) were used for directed gene delivery and HChrR6 mRNA as gene. Here, the clinical transfer of this approach is enhanced by: (i) use of CB1954 (tretazicar) for which safe human dose is established; HChrR6 can activate this prodrug. (ii) EVs delivered in vitro transcribed (IVT) HChrR6 mRNA, eliminating the potentially harmful plasmid transfection of EV producer cells we utilized previously; this has not been done before. IVT mRNA loading of EVs required several steps. Naked mRNA being unstable, we ensured its prodrug activating functionality at each step. This was not possible using tretazicar itself; we relied instead on HChrR6's ability to convert CNOB into MCHB, whose fluorescence is easily visualizable. HChrR6 mRNA-translated product's ability to generate fluorescence from CNOB vicariously indicated its competence for tretazicar activation. (iii) Systemic IVT mRNA-loaded EVs displaying an anti-HER2 single-chain variable fragment ("IVT EXO-DEPTs") and tretazicar caused growth arrest of human HER2+ breast cancer xenografts in athymic mice. As this occurred without injury to other tissues, absence of off-target mRNA delivery is strongly indicated. Many cancer sites are not amenable for direct gene injection, but current GDEPTs require this. In circumventing this need, a major advance in GDEPT applicability has been accomplished.
Subject(s)
Bacterial Proteins/genetics , Breast Neoplasms/therapy , Extracellular Vesicles/metabolism , Gene Transfer Techniques , Genetic Therapy , Prodrugs/pharmacology , RNA, Messenger/administration & dosage , Animals , Apoptosis , Bacterial Proteins/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Proliferation , Extracellular Vesicles/genetics , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptor, ErbB-2/metabolism , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
This paper deals with specific targeting of the prodrug/enzyme regimen, CNOB/HChrR6, to treat a serious disease, namely HER2+ human breast cancer with minimal off-target toxicity. HChrR6 is an improved bacterial enzyme that converts CNOB into the cytotoxic drug MCHB. Extracellular vesicles (EV) were used for mRNA-based HchrR6 gene delivery: EVs may cause minimal immune rejection, and mRNA may be superior to DNA for gene delivery. To confine HChrR6 generation and CNOB activation to the cancer, the EVHB chimeric protein was constructed. It contains high-affinity anti-HER2 scFv antibody (ML39) and is capable of latching on to EV surface. Cells transfected with EVHB-encoding plasmid generated EVs displaying this protein ("directed EVs"). Transfection of a separate batch of cells with the new plasmid, XPort/HChrR6, generated EVs containing HChrR6 mRNA; incubation with pure EVHB enabled these to target the HER2 receptor, generating "EXO-DEPT" EVs. EXO-DEPT treatment specifically enabled HER2-overexpressing BT474 cells to convert CNOB into MCHB in actinomycin D-independent manner, showing successful and specific delivery of HChrR6 mRNA. EXO-DEPTs-but not undirected EVs-plus CNOB caused near-complete growth arrest of orthotopic BT474 xenografts in vivo, demonstrating for the first time EV-mediated delivery of functional exogenous mRNA to tumors. EXO-DEPTs may be generated from patients' own dendritic cells to evade immune rejection, and without plasmids and their potentially harmful genetic material, raising the prospect of clinical use of this regimen. This approach can be used to treat any disease overexpressing a specific marker. Mol Cancer Ther; 17(5); 1133-42. ©2018 AACR.
