Thursday, November 21, 2024
11/21/2024
Project Summary The goal of this proposal is to continue the development of a synthetic live bacterial therapeutic for homocystinuria, an inborn metabolic disorder leading to accumulation of the amino acid methionine, and results in dramatically increased risk of stroke and other thrombotic conditions. Petri Bio’s approach will be capable of breaking down methionine in the gut to reduce or eliminate dependence on a methionine restricted diet and result in decreased plasma and tissue homocysteine resulting in superior clinical outcomes. The condition is estimated to occur at an incidence of 1 in 250,000, however some reports indicate a potential incidence of 1 in 65,000 when accounting for the current imprecise diagnostic assays and often subtle symptoms which may evade clinical detection until they become severe or obvious, such as lens detachment from the center of the eye. Cystathionine beta-synthase (CBS), the gene mutated in classical homocystinuria, is localized at a key regulatory branch point in the eukaryotic methionine cycle. CBS catalyzes a pyridoxal 5′-phosphate dependent beta-replacement reaction condensing serine and homocysteine (Hcy) into cystathionine that is subsequently converted to cysteine in a reaction catalyzed by cystathionine-γ-lyase (CGL, EC 4.4.1.1). Inactivation of CBS by mutation results in classical homocystinuria (HCU) which in human subjects, is characterized by a range of connective tissue disturbances including marfanoid habitus and lens dislocation, intellectual impairment, and a dramatically increased incidence of vascular disorders particularly thromboembolic complications such as stroke. Treatment strategies for pyridoxine non-responsive HCU typically attempt to lower plasma and tissue levels of Hcy by a combination of restricting dietary intake of the Hcy precursor methionine and dietary supplementation with trimethylglycine, more commonly referred to as betaine. Petri Bio, Inc. has developed a novel strategy for enzyme therapy, employing prokaryotic strains compatible with the human gut microbiome to serve as expression vectors for therapeutic protein administration. After in silico screening of bacterially-derived methionases for a number of desirable characteristics of therapeutic enzymes, ten have been cloned, expressed, and shown to reduce methionine concentrations in vitro. During this Phase I program, we will extend these studies by screening hundreds of bacterially-derived methionases in silico and subsequently cloning, expressing, and testing in vitro methionine catalysis capabilities. In vitro tests will be undertaken to measure methionase activity of these bacterial strains. After optimization of strains with high methionase activity, we will evaluate their ability to reduce methionine concentrations in vivo, as well as ameliorate the effects of methionine accumulation in a murine model of HCU. Future studies will optimize the bacterial methionase transgenes to ensure maximum activity and biocompatibility as well as select a lead candidate bacterial strain for preclinical drug development.
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