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.

中文翻译：

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细胞外囊泡介导的体外转录 mRNA 递送用于通过前药 CB1954 治疗小鼠 HER2+ 乳腺癌异种移植物，无一般毒性

前药在被细菌或病毒基因产物激活之前是无害的；它们构成了称为 GDEPT 的基因传递前药疗法的基础，该疗法可以杀死肿瘤而不会产生重大副作用。以前，我们利用 GDEPT 中的前药 CNOB（C16H7CIN2O4；未经过临床测试）和酶 HChrR6 在肿瘤中生成药物 MCHB（C16H9CIN2O2）。细胞外囊泡 (EV) 用于定向基因递送，HChrR6 mRNA 作为基因。在这里，这种方法的临床转移通过：(i) 使用 CB1954（tretazicar）确定了安全的人用剂量；HChrR6 可以激活这种前药。(ii) EVs 提供体外转录 (IVT) HChrR6 mRNA，消除了我们之前使用的 EV 生产细胞的潜在有害质粒转染；以前没有这样做过。EV 的 IVT mRNA 加载需要几个步骤。裸 mRNA 不稳定，我们确保其在每一步都具有前药激活功能。使用 tretazicar 本身是不可能的；相反，我们依靠 HChrR6 将 CNOB 转化为 MCHB 的能力，其荧光很容易可视化。HChrR6 mRNA 翻译产物从 CNOB 产生荧光的能力间接表明其具有激活tretazicar 的能力。(iii) 显示抗 HER2 单链可变片段（“IVT EXO-DEPTs”）的全身性 IVT mRNA 加载的 EVs 和曲他他卡导致无胸腺小鼠中人 HER2+ 乳腺癌异种移植物的生长停滞。由于这不会对其他组织造成伤害，因此强烈表明没有脱靶 mRNA 递送。许多癌症部位不适合直接基因注射，但当前的 GDEPT 需要这样做。为了规避这种需要，