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.

Fig 1. Trans-cinnamate production by cell line.

Trans-cinnamate produced as a result of phe catabolism by vector control (pSLERGT) or pHENOMMenal (pHENOMM) cell lysates when held at 37°C. Time points 5, 10, 15, 20, 25, and 30 minutes showed significant production of trans-cinnamate in pHENOMMenal cell lysate compared to the control pSLERGT lysate (P < 0.05, 0.001, 0.0005, 0.001, 0.00025, and 0.0001 respectively). Each data point represents the mean of triplicate wells at 5 minute intervals starting at 0 minutes and ending at 30 minutes. Error bars indicate standard deviation. (Trans-cinnamate standard curve inset).

Fig 2. Preliminary efficacy of ingested probiotics.

Blood samples were collected from PKU mice prior to treatment, and samples were collected from the same animals after treatment. Each dot and tie line represents one animal. (A) PKU male mice were fed control probiotic for four days prior to second sample collection. Data shown are results pooled from two independent experimental runs. P > 0.25. (B) PKU mice were fed pHENOMMenal probiotic for four days (males, circles) or three days (females, triangles) prior to second sample collection. Data shown are pooled results from three independent experimental runs, P < 0.0005.

Fig 3. Effect of seven day pHENOMMenal treatment on blood phe levels.

(A) Blood was collected prior to and after PKU mice were fed pHENOMMenal probiotic for seven days. Each dot connected by a line indicates an individual mouse (P < 1.0⁻⁵, n = 12). Data is a pool of four independent experiments, three experiments used male animals and one experiment used female animals. (B) Blood was collected prior to treatment, after seven days of treatment, and again after fourteen days of treatment with pHENOMMenal probiotic. Significant reduction in blood phe compared to the control is observed at seven days and fourteen days (P < 0.025 and P < 0.005 respectively), but no significant reduction is observed between day seven and day fourteen (P > 0.25). Data representative of four animals (n = 4), numbers correspond to the same animal in the graph for 3A.

Fig 4. Anti-AvPAL antibody titers.

(A) Anti-AvPAL IgG titers from animals injected on days zero, seven, and fourteen with 0μg, 30μg, or 150μg AvPAL. (B) anti-AvPAL IgG titers from each mouse in Fig 4 throughout and after the fourteen day treatment study. Collections shown from left to right for each animal are prior to treatment, after fourteen days of pHENOMMenal treatment, and four months after cessation of treatment