Improvements of SYNB1353, an Engineered Bacteria for the Treatment of Homocystinuria, Lead to Increased in Vitro and In Vivo Degradation of Methionine.

Classic homocystinuria (HCU) is an inherited disorder caused by mutations in the cystathionine beta-synthase gene resulting in excessive accumulation of the methionine (Met) degradation product homocysteine (Hcy) and multiorgan clinical manifestations. Early initiation of methionine-restricted diets in HCU patients significantly lowers the risk of developing complications from HCU. To replicate the benefits of Met restriction, the probiotic Escherichia coli Nissle 1917 was engineered to generate SYNB1353, a strain designed to degrade Met in the gut and prevent its absorption and conversion to Hcy. SYNB1353 was engineered to facilitate the intake of gastrointestinal Met via a Met importer (MetP) and to convert Met into 3-methylthiopropylamine (3-MTP) via the pyridoxal-5-phosphate (PLP)-dependent Met decarboxylase (MetDC, Q70D N82H). SYNB1353 was evaluated in healthy volunteers and was well-tolerated. Administration of an oral bolus of Met in these subjects resulted in transient elevations of plasma Met and Hcy, which were blunted by SYNB1353 compared to placebo. In preparation for Phase 2 in HCU patients, manufacturing process development continues to improve strain activity. A 2-fold in vitro improvement was evaluated in mice treated with d₄-methionine, in which this new fermentation process resulted in increased urinary 3-MTP biomarker levels and significant decrease of plasma d₄-methionine (p <0.05) compared to the old process. Proteomics of SYNB1353 produced with different fermentation processes revealed the presence of a potential PLP competitor that could impact MetDC activity which can be repressed using fermentation conditions. Additional work is ongoing to optimize methionine degradation and SYNB1353’s potential for treatment of HCU.