Programmable encapsulation systems to improve delivery of therapeutic bacteria

项目总结/摘要 癌症免疫疗法的最新进展为患有三重癌症的患者提供了有希望的治疗选择。 阴性乳腺癌（TNBC）。尽管在治疗这些恶性肿瘤方面取得了总体成功，但免疫组化- 这些方法面临着许多独特的挑战：（1）由于脱靶副作用导致的剂量限制，（2）附加毒性 联合治疗，（3）乳腺癌的免疫原性相对较低。为了克服这些局限性， 该提案寻求设计益生菌菌株，选择性地在乳腺癌肿瘤中定植 来源和局部释放治疗剂。最终目标是创造出临床上可用的菌株， 释放高剂量的治疗药物，同时保持患者的安全性。 为了做到这一点，伴随的项目将集中在工程遗传编码封装系统， 用于乳腺癌治疗治疗性细菌的改进的静脉内（IV）递送。在以前的研究中，我们 发现单次注射益生菌菌株E.大肠杆菌Nissle 1917（EcN）可以在多种原发性和 乳腺来源的转移性肿瘤，与具有跨器官转移性疾病的TNBC患者相关， 如肝、脑、骨和肺。然而，在静脉注射遗传减毒细菌的临床试验中， 不到15%的患者表现出有效的肿瘤定殖，尽管安全的给药剂量 知道了使用合成生物学方法，我们先前设计了EcN，用于瞬时诱导胶囊 细菌表面的多糖（称为iCAP），通过以下方式增加小鼠的最大耐受剂量： 十倍在第一个目标中，我们将在基因组学上整合该系统，并将其与所使用的遗传衰减联合收割机相结合。 在临床试验中，以及我们建立的其他安全系统中。我们将去除益生菌中的抗生素标记 也是为了将来的翻译。由于对我们的EcN-iCAP的体液抗体应答将在 对于人类，它们将限制系统产生多次注射的功效。接下来，我们将构建两个 其他包封系统（聚唾液酸、软骨素），以使得能够顺序递送不同的菌株。这些 这些方法将通过多种体外试验和小鼠模型进行表征和测试。 总的来说，使用工程益生菌的方法比目前的治疗策略有几个优点， 包括：（1）治疗剂的肿瘤特异性生产，（2）导致有效释放的细菌裂解， 新的治疗剂和脂多糖（LPS）佐剂，和（3）增强的联合治疗的功效和安全性， 国家和多个封装系统。这项工作旨在克服目前免疫疗法的局限性， Pies，通过提供靶向载体来局部递送刺激抗肿瘤免疫的治疗剂，同时预 释放全身毒性并减轻免疫相关的不良反应。

PROJECT SUMMARY/ABSTRACT Recent advances in cancer immunotherapy have provided promising treatment options for patients with triple- negative breast cancer (TNBC). Despite overall success in treating these malignancies, immunotherapeutic ap- proaches face a number of unique challenges: (1) dose limitation due to off-target side effects, (2) additive toxicity of combination therapies, (3) and relatively low immunogenicity of breast cancer. To overcome these limitations, this proposal seeks to engineer probiotic strains of bacteria that selectively colonize tumors of breast-cancer origin and locally release therapeutics. The ultimate goal is to create clinic-ready strains that will efficiently local- ize and release high-doses of therapeutics, while maintaining safety for patients. To do so, the accompanying project will focus on engineering genetically encoded encapsulation systems to improve intravenous (IV) delivery of therapeutic bacteria for breast cancer therapy. In previous studies, we have found that a single injection of a probiotic strain E. coli Nissle 1917 (EcN) can colonize multiple primary and metastatic tumors of breast origin, relevant for TNBC patients that have metastatic disease across organs such as the liver, brain, bone, and lung. However, in clinical trials with IV injection of genetically-attenuated bacteria, less than 15% of patients demonstrated efficient colonization of tumors, although safe administration doses were noted. Using synthetic biology approaches, we previously engineered EcN for transient induction of capsule polysaccharides on the bacteria surface (termed iCAP), which increases maximally-tolerated doses in mice by 10-fold. In the first aim, we will genomically integrate this system and combine it with genetic attenuations used in clinical trials, and other safety systems we have built. We will remove antibiotic markers from the probiotic strain for future translation as well. Since humoral antibody responses to our EcN-iCAP will be generated in humans, they will limit the efficacy of the system to generate multiple injections. Thus, we will next construct two other encapsulation systems (polysialic acid, chondroitin), to enable sequential delivery of distinct strains. These approaches will be characterized and tested through multiple in vitro assays and in mouse models. Altogether the approach of using engineered probiotics has several advantages over current therapeutic strat- egies, including: (1) tumor-specific production of therapeutics, (2) bacteria lysis that leads to effective release of novel therapeutics and lipopolysaccharides (LPS) adjuvant, and (3) enhanced efficacy and safety from combi- nations and multiple encapsulation systems. This work seeks to overcome current limitations of immunothera- pies, by providing a targeted vehicle to locally deliver therapeutics that stimulate antitumor immunity while pre- venting systemic toxicity and mitigating immune-related adverse effects.