Institute for Molecular Imaging and Theranostics, Chonnam National University Medical School, Gwangju, 61469, Republic of Korea.
Department of Molecular Medicine (BrainKorea21 Plus), Chonnam National University Graduate School, Gwangju, 61469, Republic of Korea.
Mol Imaging Biol. 2022 Feb;24(1):82-92. doi: 10.1007/s11307-021-01624-x. Epub 2021 Aug 17.
In the programming of tumor-targeting bacteria, various therapeutic or reporter genes are expressed by different gene-triggering strategies. Previously, we engineered pJL87 plasmid with an inducible bacterial drug delivery system that simultaneously co-expressed two genes for therapy and imaging by a bidirectional tet promoter system only in response to the administration of exogenous doxycycline (Doxy). In this multi-cassette expression approach, tetA promoter (P) was 100-fold higher in expression strength than tetR promoter (P). In the present study, we developed pJH18 plasmid with novel Doxy-inducible gene expression system based on a tet promoter.
In this system, Tet repressor (TetR) expressed by a weak constitutive promoter binds to tetO operator, resulting in the tight repression of gene expressions by P and P, and Doxy releases TetR from tetO to de-repress P and P.
In Salmonella transformed with pJH18, the expression balance of bidirectional tet promoters in pJH18 was remarkably improved (P:P = 4~6:1) compared with that of pJL87 (P:P = 100:1) in the presence of Doxy. Also, the expression level by novel tet system was much higher in Salmonella transformed with pJH18 than in those with pJL87 (80-fold in rluc8 and 5-fold in clyA). Interestingly, pJH18 of the transformed Salmonella was much more stably maintained than pJL87 in antibiotic-free tumor-bearing mice (about 41-fold), because only pJH18 carries bom sequence with an essential role in preventing the plasmid-free population of programmed Salmonella from undergoing cell division.
Overall, doxycycline-induced co-expression of two proteins at similar expression levels, we exploited bioluminescence reporter proteins with preclinical but no clinical utility. Future validation with clinically compatible reporter systems, for example, suitable for radionuclide imaging, is necessary to develop this system further towards potential clinical application.
在肿瘤靶向细菌的编程中，各种治疗或报告基因通过不同的基因触发策略表达。此前，我们通过双向 tet 启动子系统设计了 pJL87 质粒，该系统可诱导细菌药物输送系统，仅在外源多西环素（Doxy）给药时同时共表达两种用于治疗和成像的基因。在这种多盒表达方法中，tetA 启动子（P）的表达强度比 tetR 启动子（P）高 100 倍。在本研究中，我们开发了基于 tet 启动子的新型 Doxy 诱导基因表达系统的 pJH18 质粒。
在该系统中，由弱组成型启动子表达的 Tet 阻遏物（TetR）与 tetO 操纵子结合，导致 P 和 P 的基因表达受到紧密抑制，而 Doxy 从 tetO 释放 TetR 以解除 P 和 P 的抑制。
在转化为 pJH18 的沙门氏菌中，与存在 Doxy 时的 pJL87 相比（P:P=100:1），pJH18 中双向 tet 启动子的表达平衡得到显著改善（P:P=4~6:1）。此外，与 pJL87 相比（在 rluc8 中为 80 倍，在 clyA 中为 5 倍），新型 tet 系统的表达水平在转化为 pJH18 的沙门氏菌中要高得多。有趣的是，与抗生素-free 荷瘤小鼠中的 pJL87 相比，转化为 pJH18 的沙门氏菌的质粒保持得更稳定（约 41 倍），因为只有 pJH18 携带 bom 序列，该序列对于防止编程沙门氏菌的无质粒种群进行细胞分裂至关重要。
总的来说，我们利用了具有临床前但无临床用途的生物发光报告蛋白，实现了两种蛋白质在类似表达水平下的 Doxy 诱导共表达。未来需要与临床兼容的报告系统（例如适用于放射性核素成像的报告系统）进行验证，以进一步开发该系统，使其更有可能应用于临床。
