Data_Sheet_1_Challenges and Pitfalls in the Engineering of Human Interleukin 22 (hIL-22) Secreting Lactobacillus reuteri.pdf

Engineered microbes for the delivery of intestinally directed therapeutics is a promising avenue for the treatment of various intestinal diseases including inflammatory bowel disease (IBD) and intestinal graft vs. host disease (GVHD). This modality of treatment would allow for the targeted delivery of therapeutics to the site of inflammation or disease while minimizing the systemic side effects that often accompany treatment of these pathologies. Here, we show the challenges encountered and overcome in successfully engineering Lactobacillus reuteri to secrete high levels of biologically active human interleukin 22 (hIL-22). Initial hIL-22 constructs secreted high levels of hIL-22, however we found the majority of hIL-22 was cleaved and not biologically active. Several strategies were explored to improve the production of intact hIL-22, with the optimization of the signal sequence for peptide secretion having the most impact of production of intact hIL-22. This resulted in L. reuteri secreting high concentrations (up to 700 ng/mL) of hIL-22. Bioactivity of hIL-22 was confirmed by the secretion of interleukin 10 (IL-10) from the colon cancer derived epithelial cell line Colo205 and the secretion of Regenerating islet-derived protein 3 alpha (Reg3α) from human jejunal enteroids. The secretion of bioactive hIL-22 imposed a significant cost for L. reuteri as bacterial growth was significantly impaired upon induction. Future challenges and optimization strategies for the delivery of hIL-22 to the human intestinal tract are discussed.

用于肠道靶向递送治疗制剂的工程化微生物（Engineered microbes for the delivery of intestinally directed therapeutics）是治疗包括炎症性肠病（inflammatory bowel disease, IBD）、肠道移植物抗宿主病（intestinal graft vs. host disease, GVHD）在内的多种肠道疾病的极具潜力的策略。该治疗模式可实现治疗剂向炎症或病变部位的靶向递送，同时最大限度降低此类疾病治疗中常伴随的全身性不良反应。本研究阐明了在成功构建可分泌高活性人白细胞介素22（human interleukin 22, hIL-22）的罗伊氏乳杆菌（Lactobacillus reuteri）过程中遇到并得以攻克的各类难题。初始构建的hIL-22表达构建体可分泌大量hIL-22，但我们发现绝大多数hIL-22发生了蛋白水解切割，丧失了生物活性。本研究探索了多种提升完整hIL-22产量的策略，其中优化用于肽类分泌的信号序列（signal sequence）对完整hIL-22的产量提升效果最为显著。该优化方案使罗伊氏乳杆菌可分泌高达700 ng/mL的hIL-22。通过结肠癌来源上皮细胞系Colo205分泌白细胞介素10（interleukin 10, IL-10），以及人空肠类器官（human jejunal enteroids）分泌胰岛再生源蛋白3α（Regenerating islet-derived protein 3 alpha, Reg3α），验证了hIL-22的生物活性。但分泌具有生物活性的hIL-22会对罗伊氏乳杆菌造成显著的代谢负荷：诱导表达后，细菌的生长受到明显抑制。本研究最后讨论了将hIL-22递送至人体肠道所面临的后续挑战及优化策略。