Abstract
PurposeIn 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 (PtetA) was 100-fold higher in expression strength than tetR promoter (PtetR). In the present study, we developed pJH18 plasmid with novel Doxy-inducible gene expression system based on a tet promoter.ProceduresIn this system, Tet repressor (TetR) expressed by a weak constitutive promoter binds to tetO operator, resulting in the tight repression of gene expressions by PtetA and PtetR, and Doxy releases TetR from tetO to de-repress PtetA and PtetR.ResultsIn Salmonella transformed with pJH18, the expression balance of bidirectional tet promoters in pJH18 was remarkably improved (PtetA:PtetR = 4~6:1) compared with that of pJL87 (PtetA:PtetR = 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.ConclusionsOverall, 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.
Highlights
The disordered tumor microenvironment is responsible for oxygen deficiency, excessive nutrient leakage, and immune privilege, which can provide a niche in which bacteria such as Clostridium [1], Bifidobacterium [2, 3], Listeria [4, 5], Escherichia coli [6, 7], and Salmonella [8, 9] can survive
We previously developed an attenuated strain of Salmonella typhimurium that was defective in guanosine 5′-diphosphate-3′-diphosphate synthesis (ΔppGpp Salmonella, SAM) and which showed preferential accumulation in tumors, resulting in bacterial numbers that were more than 10,000-fold higher in tumor tissue than in healthy organs [12]
This report describes our engineering of a novel Doxyinducible gene expression system for bacteria-based cancer therapy in which multiple proteins are expressed in a balanced way by a single vector containing the bidirectional promoters PtetA and PtetR
Summary
The disordered tumor microenvironment is responsible for oxygen deficiency, excessive nutrient leakage, and immune privilege, which can provide a niche in which bacteria such as Clostridium [1], Bifidobacterium [2, 3], Listeria [4, 5], Escherichia coli [6, 7], and Salmonella [8, 9] can survive. Bacteria are well-suited for distinguishing tumor tissue from normal organs because they colonize tumors and proliferate within them [10, 11]. We previously developed an attenuated strain of Salmonella typhimurium that was defective in guanosine 5′-diphosphate-3′-diphosphate synthesis (ΔppGpp Salmonella, SAM) and which showed preferential accumulation in tumors, resulting in bacterial numbers that were more than 10,000-fold higher in tumor tissue than in healthy organs [12]. As attenuated bacteria alone are often unable to eradicate malignant tumors, various bacterial species have been subjected to genetic programming to develop tumorselective protein–drug factories [8, 9, 12–15]. Because bacteria tend to localize initially, but transiently, in the liver and spleen [11, 16], various inducible effector systems that utilize external gene triggers such as Larabinose [8, 9, 12, 17], salicylate [18], γ-irradiation [19], and tetracycline [8] have been actively developed to maximize intratumoral effects while minimizing systemic toxicity
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