Engineering Salmonella to be a targeted intratumoral anti-cancer therapeutic

描述(申请人提供)：将鼠伤寒沙门氏菌改造为靶向抗癌治疗细菌，专门针对肿瘤的治疗耐药区域，并可控制地杀死癌细胞，将能够克服治疗耐药，提高癌症治疗的有效性。治疗耐药性是由于药物渗透率有限和细胞敏感性差造成的。只有能够穿透肿瘤组织并克服扩散限制的运动型细菌才能有效地杀死远离肿瘤血管的耐药细胞。因此，控制细菌的运动能力是开发能够克服治疗耐药性的有效疗法的关键。到目前为止，细菌在肿瘤中的运动机制还知之甚少。我们的方法将阐明这些机制，并将操纵它们以肿瘤静止为靶点。该项目是独一无二的，因为它引入了肿瘤内治疗提供的概念和圆柱形肿瘤模型，该模型专门设计用于量化肿瘤中的细菌趋化性。拟议的研究计划有三个具体目标，结合了肿瘤生物学、分子生物学和数学建模方面的专业知识。它们是相互关联的，并将同时进行：目标1是确定控制鼠伤寒沙门氏菌在肿瘤中聚集的机制；目标2是设计具有增强靶向性的鼠伤寒沙门氏菌突变体；目标3是创造具有更高肿瘤细胞毒性的鼠伤寒沙门氏菌突变体。研究计划的三个假设是：1)鼠伤寒沙门氏菌被肿瘤细胞吸引并诱导细胞凋亡，2)核糖受体敲除鼠伤寒沙门氏菌优先聚集在肿瘤的静止区域，3)鼠伤寒沙门氏菌表达辐射诱导的细胞毒性多肽有效载荷将更有效地减少肿瘤质量。这些假说将通过测量鼠伤寒沙门氏菌在小鼠肿瘤中的定位，从鼠伤寒沙门氏菌基因组中删除核糖受体，以及在RecA启动子的控制下转化鼠伤寒沙门氏菌表达mTRAIL多肽来验证。转化的细菌减少肿瘤质量的效率将在培养和小鼠身上进行测量。该实验计划是一项研究计划的一部分，该计划旨在制定一种克服治疗耐药性的治疗策略。联合应用抗肿瘤靶向的鼠伤寒沙门氏菌和辅助化疗将通过更有效地杀死远离肿瘤血管的癌细胞来提高治疗效率。未来的人体试验将调查联合使用细菌和化疗来减少四期乳腺癌患者局部复发和转移疾病的能力。利用细菌克服治疗耐药性，提高治疗效率，将显著降低全身毒性，限制转移性疾病的有害影响，并延长预期寿命。该实验计划描述了一种使用静止靶向、可控制的细胞毒性鼠伤寒沙门氏菌来克服治疗耐药性的治疗策略。工程菌和辅助化疗的联合应用将通过更有效地杀死肿瘤中的所有癌细胞来提高治疗效率。这种增加的疗效将减少全身毒性，防止局部复发，限制转移性疾病的有害影响，并延长预期寿命。

DESCRIPTION (provided by applicant): Engineering Salmonella typhimurium to be a targeted anti-cancer therapeutic Bacteria engineered to specifically target therapeutically resistant regions of tumors and controllably kill cancer cells will be able to overcome therapeutic resistance and increase the efficacy of cancer treatment. Therapeutic resistance is caused by limited drug penetration and poor cell susceptibility. Only motile bacteria, which can penetrate into tumor tissue and overcome diffusion limitations, are able to effectively kill therapeutically resistant cells distant from tumor vasculature. Controlling bacterial motility is therefore the key to developing effective therapies able to overcome therapeutic resistance. To date, the mechanisms of bacterial motility in tumors are poorly understood. Our approach will elucidate these mechanisms and will manipulate them to target tumor quiescence. The project is unique because it introduces the concept of intratumoral therapeutic delivery and the cylindroid tumor model, which was specifically designed to quantify bacterial chemotaxis in tumors. The proposed research program has three Specific Aims that combine expertise in tumor biology, molecular biology, and mathematical modeling. They are interrelated and will be performed concurrently: Aim 1 is to determine the mechanisms that control S. typhimurium accumulation in tumors; Aim 2 is to design S. typhimurium mutants with enhanced targeting; and Aim 3 is to create S. typhimurium mutants with increased tumor cytotoxicity. The three hypotheses of the research plan are: 1) S. typhimurium are attracted to and induce cellular apoptosis in tumors, 2) ribose-receptor-knockout S. typhimurium preferentially accumulate in quiescent regions of tumors, and 3) S. typhimurium expressing a radiation-inducible, cytotoxic polypeptide payload will more effectively reduce tumor mass. These hypotheses will be tested by measuring the localization of S. typhimurium in mouse tumors, deleting the ribose receptor from the S. typhimurium genome, and transforming S. typhimurium to express the mTRAIL polypeptide under control of the RecA promoter. The efficiency of the transformed bacteria to reduce tumor mass will be measured in culture and in mice. The experimental plan is part of a research program to develop a therapeutic strategy to overcome therapeutic resistance. Combined administration of tumor-quiescence-targeted S. typhimurium and adjuvant chemotherapy will increase therapeutic efficiency by more effectively killing cancer cells distant from tumor vasculature. Future human trials will investigate the ability of combined administration of bacteria and chemotherapy to reduce local recurrence and metastatic disease in stage-four breast cancer patients. Using bacteria to overcome therapeutic resistance and increase treatment efficiency will significantly reduce systemic toxicity, limit the deleterious effects of metastatic disease, and increase life expectancy. The experimental plan descries a therapeutic strategy to overcome therapeutic resistance using quiescence- targeted, controllably cytotoxic S. typhimurium. Combined administration of engineered bacteria and adjuvant chemotherapy will increase therapeutic efficiency by more effectively killing all cancer cells in tumors. This increased efficacy will reduce systemic toxicity, prevent local recurrence, limit the deleterious effects of metastatic disease, and increase life expectancy.