Improvement of a synthetic live bacterial therapeutic for phenylketonuria with biosensor-enabled enzyme engineering

Abstract

In phenylketonuria (PKU) patients, a genetic defect in the enzyme phenylalanine hydroxylase (PAH) leads to elevated systemic phenylalanine (Phe), which can result in severe neurological impairment. As a treatment for PKU, Escherichia coli Nissle (EcN) strain SYNB1618 was developed under Synlogic’s Synthetic Biotic™ platform to degrade Phe from within the gastrointestinal (GI) tract. This clinical-stage engineered strain expresses the Phe-metabolizing enzyme phenylalanine ammonia lyase (PAL), catalyzing the deamination of Phe to the non-toxic product trans-cinnamate (TCA). In the present work, we generate a more potent EcN-based PKU strain through optimization of whole cell PAL activity, using biosensor-based high-throughput screening of mutant PAL libraries. A lead enzyme candidate from this screen is used in the construction of SYNB1934, a chromosomally integrated strain containing the additional Phe-metabolizing and biosafety features found in SYNB1618. Head-to-head, SYNB1934 demonstrates an approximate two-fold increase in in vivo PAL activity compared to SYNB1618.

An engineered bacterial therapeutic lowers urinary oxalate in preclinical models and in silico simulations of enteric hyperoxaluria

“…Bacteria have been engineered for a wide range of therapeutic functions for the treatment of diseases including cancer, immune disorders, and for infection (Sola‐Oladokun et al , 2017 ; Drolia et al , 2020 ). The application of engineered bacteria to metabolic diseases, such as PKU, has involved engineering probiotic bacteria to eliminate toxic metabolites from the GI tract (Isabella et al , 2018 ; Charbonneau et al , 2020 ; Adolfsen et al , 2021 ). In addition to translational models that characterize in vitro and in vivo strain activity, in silico models that can be used to predict strain activity in humans have been developed (Charbonneau et al , 2021 ; Nelson et al , 2021 ).…”

Microcin MccI47 selectively inhibits enteric bacteria and reduces carbapenem-resistant Klebsiella pneumoniae colonization in vivo when administered via an engineered live biotherapeutic

“…Under the global societal challenge of moving towards more personalized medicine, biomedical research is currently exploring the use of engineered bacteria as vehicles for the targeted administration of therapeutic compounds at the location of disease and with the goal of reducing systemic side effects [33,41,42]. This is particularly relevant when the therapeutic objective is the delivery of protein-based agents (i.e., AMPs) to the GI tract as host digestive enzymes likely inactivate them before they reach their site of action [43].…”

Recommendations on phenylketonuria in Turkey

“…Recently gene therapy for PKU has been reported as a promising approach for both protection from neurocognitive problems, and providing a normal diet without Phe restriction for PKU patients. Preclinical studies using adeno-associated virus (AAV)-mediated gene addition therapy in the Pah enu2/enu2 mouse model in classical PKU 45,46 , genetically engineered probiotics for PKU treatment (phenylalanine lyase gene from Anabaena variabilis (AvPAL) in the Pah enu2/enu2 mouse model) 47 , genetically engineered live bacteria (Escherichia coli Nissle) 48,49 , gene correction therapy using CRISPR-Cas-9 associated base editors 50 have reported these investigational therapeutic approaches might be novel interventions for PKU. Gene correction therapy using CRISPR- Cas-9 associated base editors have been shown to provide >20% of the normal activity of PAH activity in Pah enu2 mice.…”

“…50 There are new Phase 1 and Phase 2 studies initiated to provide more effective therapeutic approaches in PKU by using live biotherapeutic products (LBPs) such as SYNB1618 in addition to Escherichia coli Nissle 1917 (EcN). 49 Statement #12:…”