Kady Mohamed R, Elston R Nicholas, Snyder Lauren J, Fleischman Jorie D, Brandt Madilyn J, Zhu Duolong, Britton Robert A, Beeton Christine
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA.
Medical Scientist Training Program, Baylor College of Medicine, Houston, TX, USA.
Microb Cell Fact. 2025 Aug 2;24(1):177. doi: 10.1186/s12934-025-02800-2.
Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint inflammation and damage in which fibroblast-like synoviocytes (FLS) play a central role. Invasiveness and proliferation of FLS in RA is dependent on activity of the K1.1 potassium channel. Peptide blockers of K1.1, such as iberiotoxin (IbTX), can be delivered subcutaneously to treat animal models of RA. We tested whether an engineered probiotic oral delivery platform could effectively deliver IbTX systemically in a rat model of RA.
A plasmid for inducible secretion of IbTX was constructed and transformed into probiotic Limosilactobacillus reuteri ATCC PTA-6475 to generate LrIbTX. No differences in growth rate between LrIbTX and the control strain were detected in vitro, and live LrIbTX was recovered from the feces of rats following oral gavage. IbTX was detected in the sera of healthy rats orally gavaged with LrIbTX by a K1.1 competitive binding assay using a biotinylated IbTX analog and streptavidin-conjugated fluorophore. Collagen-induced arthritis (CIA), an animal model of RA, was used to measure the effect of LrIbTX versus injected IbTX or control L. reuteri expressing an irrelevant protein on clinical score, histologic inflammation, and bone density. Oral LrIbTX and injected IbTX had similar efficacy in treating CIA in rats as measured by clinical joint swelling and histologic inflammation, which were significantly improved versus control bacteria or vehicle injection. No treatments induced measurable levels of anti-IbTX IgG, and there were no differences in macroscopic bone damage or anti-collagen II IgM and IgG levels between CIA groups. Injected IbTX and oral LrIbTX were also equivalent in inhibiting an active delayed-type hypersensitivity (DTH) reaction in rats.
This work describes the effective oral delivery of a candidate therapeutic peptide, IbTX, via engineered L. reuteri to treat an animal model of autoimmune disease and demonstrates systemic distribution of an intestinally produced peptide. We anticipate that oral delivery of engineered microbes may be a generalizable strategy for enhancing the oral bioavailability of peptide therapeutics.
类风湿性关节炎（RA）是一种自身免疫性疾病，其特征为关节炎症和损伤，其中成纤维样滑膜细胞（FLS）起核心作用。RA中FLS的侵袭性和增殖取决于K1.1钾通道的活性。K1.1的肽阻滞剂，如iberiotoxin（IbTX），可皮下给药以治疗RA动物模型。我们测试了一种工程化益生菌口服递送平台是否能在RA大鼠模型中有效全身递送IbTX。
构建了用于诱导分泌IbTX的质粒，并将其转化到益生菌罗伊氏乳杆菌Limosilactobacillus reuteri ATCC PTA - 6475中以产生LrIbTX。体外未检测到LrIbTX与对照菌株在生长速率上的差异，经口灌胃后从大鼠粪便中回收了活的LrIbTX。通过使用生物素化的IbTX类似物和链霉亲和素偶联荧光团的K1.1竞争性结合测定法，在经口灌胃LrIbTX的健康大鼠血清中检测到了IbTX。胶原诱导的关节炎（CIA），一种RA动物模型，用于测量LrIbTX与注射的IbTX或表达无关蛋白的对照罗伊氏乳杆菌相比对临床评分、组织学炎症和骨密度的影响。通过临床关节肿胀和组织学炎症测量，口服LrIbTX和注射IbTX在治疗大鼠CIA方面具有相似的疗效，与对照细菌或赋形剂注射相比有显著改善。没有治疗诱导可测量水平的抗IbTX IgG，并且CIA组之间在宏观骨损伤或抗胶原蛋白II IgM和IgG水平上没有差异。注射的IbTX和口服LrIbTX在抑制大鼠主动迟发型超敏反应（DTH）方面也等效。
这项工作描述了通过工程化的罗伊氏乳杆菌有效口服递送候选治疗肽IbTX以治疗自身免疫性疾病动物模型，并证明了肠道产生的肽的全身分布。我们预计工程化微生物的口服递送可能是提高肽治疗剂口服生物利用度的一种可推广策略。
