Tumor-targeting Salmonella expressing apoptosis-inducing cytotoxic proteins

描述(申请人提供)：与其他抗癌药物输送系统相比，肿瘤靶向细菌具有许多优势，包括从远距离接种部位靶向多个肿瘤，在肿瘤内选择性复制，高度的衰减和安全性，以及直接在肿瘤内表达抗癌蛋白的能力。值得注意的是，减毒沙门氏菌在实体肿瘤中的水平至少是其他组织的1000倍。人类临床研究已经验证了静脉注射减毒细菌突变体的安全性，建立了对多次剂量的耐受性，并表明在一些患者中发生了肿瘤靶向。然而，没有观察到抗肿瘤活性，即使在沙门氏菌被证实已在其肿瘤中定植的患者中也是如此。我们假设，人类临床试验中缺乏抗肿瘤效果的问题可以通过工程诱导凋亡(程序性细胞死亡)诱导能够杀死肿瘤细胞的细胞毒蛋白来克服。这些特定的目的是为了测试单个蛋白质和蛋白质组合，以开发具有增强抗肿瘤活性的肿瘤靶向沙门氏菌载体。该项目利用合成生物学，即DNA的化学合成，构建编码蛋白质的基因，这些蛋白质将杀死癌细胞。这些合成蛋白质的组成是基于已知的杀癌和抗癌蛋白质成分的组合，当这些成分结合在一起时，会产生具有选择性杀癌能力的蛋白质。此外，还将对细胞凋亡和细胞毒性多肽的新型双功能融合进行初步研究，以便将它们的细胞靶向、亚细胞转运和细胞内分子靶标结合起来，作为一种增强效力和肿瘤特异性的手段，并为新的拨款申请产生初步数据。这里描述的细菌非常安全，适合学生在实验室工作。学生是从PI教授的微生物学课程中招募的，并整合了他对肿瘤靶标沙门氏菌的研究。CSUN的研究生院招生计划以及MARC和RISE计划也会招收学生。尽管PI在CSUN只有一年半的时间，但他已经为11名学生提供了基于实验室的研究培训，其中包括3名西班牙裔、2名亚美尼亚人、2名印度人、1名菲律宾人、1名泰国人和1名韩国人。该项目预计将有两名研究生在两到三年的时间里攻读硕士学位，在资助期间总共有四名研究生，每年有一到两名定向研究本科生；在资助期间，每年最多有八名学生，以及每学期有一到两名学生参加生物学研究课程，在资助期间，总共有14到28名学生。

DESCRIPTION (provided by applicant): Tumor-targeting bacteria offer a number of advantages over other cancer drug delivery systems, including targeting of multiple tumors from a distant inoculation site, selective intratumoral replication, a high degree of attenuation and safety, and the ability to express anti-cancer proteins directly within the tumor. Remarkably, attenuated Salmonella localize within solid tumors at levels at least 1000 times greater than other tissues. Human clinical studies have validated the safety of intravenously administered attenuated bacterial mutants, established tolerated multiple doses, and have shown that tumor targeting occurs in some patients. However, no anti-tumor activity was observed, even in patients in which the Salmonella were verified to have colonized their tumors. We hypothesize that the lack of antitumor efficacy in the human clinical trials can be overcome by engineering apoptosis (programmed cell death) -inducing cytotoxic proteins that are able to kill tumor cells. The Specific Aims are designed to test individual proteins and protein combinations in order to develop tumor-targeted Salmonella vectors with enhanced antitumor activity. The project utilizes synthetic biology, the chemical synthesis of DNA, to construct genes encoding proteins that will kill cancer cells. The composition of these synthetic proteins is based on combinations of known cancer-killing and cancer-targeting protein components, which, when combined, result in proteins with the selective ability to kill cancer. A preliminary study of novel bifunctional fusios of the apoptosis and cytotoxic polypeptides will also be conducted in order to combine aspects of their cell targeting, subcellular transport and intracellular molecular targets as a means to confer enhanced potency together with tumor specificity, and to generate preliminary data for a new grant submission. The bacteria described here are highly safe and suitable for students to work on in the lab. Students are recruited from the microbiology courses the PI teaches and integrates his research on tumor-targeted Salmonella. Students are also recruited from the Graduate School Admission Program, and the MARC and RISE programs at CSUN. Although the PI has only been at CSUN for 1.5 years, he has already provided laboratory-based research training for 11 students, including 3 Hispanics, 2 Armenians, 2 Indians, 1 Filipino, 1 Thai, and 1 Korean. The project is expected to involve two graduate students seeking their Masters Degree over a two to three year period, with a total of up to four graduate students during the funding period, and one to two directed research undergraduates per year; up to eight during the course of the funding period, as well as one to two students per semester enrolled in the Biology research course, for a total of between 14 and 28 students during the funding period.