Genetically engineered probiotic for the treatment of phenylketonuria (PKU); assessment of a novel treatment in vitro and in the PAHenu2 mouse model of PKU

Abstract

Phenylketonuria (PKU) is a genetic disease characterized by the inability to convert dietary phenylalanine to tyrosine by phenylalanine hydroxylase. Given the importance of gut microbes in digestion, a genetically engineered microbe could potentially degrade some ingested phenylalanine from the diet prior to absorption. To test this, a phenylalanine lyase gene from Anabaena variabilis (AvPAL) was codon-optimized and cloned into a shuttle vector for expression in Lactobacillus reuteri 100-23C (pHENOMMenal). Functional expression of AvPAL was determined in vitro, and subsequently tested in vivo in homozygous PAHenu2 (PKU model) mice. Initial trials of two PAHenu2 homozygous (PKU) mice defined conditions for freeze-drying and delivery of bacteria. Animals showed reduced blood phe within three to four days of treatment with pHENOMMenal probiotic, and blood phe concentrations remained significantly reduced (P < 0.0005) compared to untreated controls during the course of experiments. Although pHENOMMenal probiotic could be cultured from fecal samples at four months post treatment, it could no longer be cultivated from feces at eight months post treatment, indicating eventual loss of the microbe from the gut. Preliminary screens during experimentation found no immune response to AvPAL. Collectively these studies provide data for the use of a genetically engineered probiotic as a potential treatment for PKU.

Adaptive Strategies of the Candidate Probiotic E. coli Nissle in the Mammalian Gut

“…Probiotics are live microorganisms that provide health benefits when consumed, including enhancement of gut epithelial barrier function, blocking of pathogen binding, and vitamin synthesis (Hill et al, 2014). There has been increasing success in expanding the therapeutic scope and efficacy of probiotics through genetic engineering, with prior work demonstrating preclinical efficacy against infectious and metabolic diseases (Durrer et al, 2017;Hwang et al, 2017;Isabella et al, 2018;Palmer et al, 2018). Engineered probiotics are exciting platforms for in situ drug synthesis and delivery, provided they maintain appropriate abundance and activity at their target site.…”

Engineering probiotics as living diagnostics and therapeutics for improving human health

“…In another study, the metabolically engineered Lactobacillus reuteri carrying PAL from Anabaena variabilis could greatly reduce blood phenylalanine in the mice model of PKU within 4-5 days of treatment. It also observed that the probiotic microbe was lost from the intestine at 8 months post treatment [43]. Recently, an engineered probiotic SYNB1618 was constructed for degradation of phenylalanine.…”

The Virtual Metabolic Human database: integrating human and gut microbiome metabolism with nutrition and disease

“…Although we could not find evidence for probiotic use in orotic aciduria, guidelines for the management of methylmalonic and propionic acidemia included the use of probiotics 57 . Furthermore, researchers have demonstrated the benefit of engineered L. reuteri strains in a murine phenylketonuria (PKU) model 58 .…”