Live microbial therapeutics for enzyme replacement therapy against homocystinuria - 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.
