Modification of an effector strain for replacement therapy of dental caries to enable clinical safety trials
Modification of an effector strain for replacement therapy of dental caries to enable clinical safety trials
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DOI：
10.1111/j.1365-2672.2007.03316.x
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发表时间：
2007-05-01
影响因子：
4
通讯作者：
Hillman, C. H.
中科院分区：
文献类型：
--
作者：
Hillman, J. D.;Mo, J.;Hillman, C. H.
目的：构建用于预防龋齿的转基因变异链球菌菌株。由于删除了乳酸脱氢酶的整个开放阅读框，该菌株显着降低了致龋性，并通过生产一种名为 mutacin 1140 的天然抗生素而具有出色的定植潜力。为了用于人体临床试验，引入了额外的突变，以便在出现不良副作用时能够快速消除该菌株，并提高遗传稳定性。方法：将缺失突变引入 dal 基因 用于 D-丙氨酸生物合成和用于遗传转化的 comE 基因。测试了所得菌株 A2JM 对外源 D-丙氨酸的依赖性及其从定植大鼠体内根除的能力。还在体外和体内模型中测试了该菌株与另一种变异链球菌菌株交换 DNA 的能力。结论：A2JM 完全依赖于外源 D-丙氨酸，但在缺乏膳食 D-丙氨酸的情况下可以在大鼠口腔中少量定殖。结果表明，A2JM 可以清除其他菌斑细菌中的 D-丙氨酸。通过每天使用氯己定降低口腔总细菌负荷，几乎可以完全根除 A2JM。 comE基因的引入并没有显着降低A2JM在体外或体内模型中的转化能力。尽管如此，在 comE 基因中添加缺失确实提供了额外的安全性，因为它具有非常低的回复频率。 研究的意义和影响：根据体外和动物模型中建立的安全性和有效性概况，A2JM 似乎适合在人体临床试验中安全使用。
Aims: To construct a genetically modified strain of Streptococcus mutans for dental caries prevention. The strain has significantly reduced cariogenicity owing to a deletion of the entire open reading frame for lactate dehydrogenase, and has excellent colonization potential through the production of a natural antibiotic called mutacin 1140. For use in human clinical trials, additional mutations were introduced to enable rapid elimination of the strain in case of adverse side effects and to increase genetic stability.Methods: Deletion mutations were introduced into the dal gene for D-alanine biosynthesis and the comE gene for genetic transformation. The resulting strain, A2JM, was tested for dependence on exogenous D-alanine and its ability to be eradicated from colonized rats. The strain was also tested for its ability to exchange DNA with another strain of S. mutans in in vitro and in vivo models.Conclusions: A2JM was completely dependent on exogenous D-alanine, but could colonize the oral cavity of rats in low numbers in the absence of dietary D-alanine. Results indicated that A2JM can scavenge D-alanine from other plaque bacteria. Lowering of the total oral bacterial load through daily application of chlorhexidine enabled virtually complete eradication of A2JM. The introduction of the comE gene did not significantly decrease the transformability of A2JM in in vitro or in vivo models. The addition of a deletion in the comE gene does, nonetheless, provide additional safety as it has a very low reversion frequency.Significance and Impact of the Study: Based on the safety and efficacy profiles established in vitro and in animal models, A2JM appears suitable for safe use in human clinical trials.