Phenylketonuria (PKU) is a genetic disease that is characterized by an inability to metabolize phenylalanine (Phe), which can result in neurotoxicity. To provide a potential alternative to a protein-restricted diet, we engineered Escherichia coli Nissle to express genes encoding Phe-metabolizing enzymes in response to anoxic conditions in the mammalian gut. Administration of our synthetic strain, SYNB1618, to the Pahenu2/enu2 PKU mouse model reduced blood Phe concentration by 38% compared with the control, independent of dietary protein intake. In healthy Cynomolgus monkeys, we found that SYNB1618 inhibited increases in serum Phe after an oral Phe dietary challenge. In mice and primates, Phe was converted to trans-cinnamate by SYNB1618, quantitatively metabolized by the host to hippurate and excreted in the urine, acting as a predictive biomarker for strain activity. SYNB1618 was detectable in murine or primate feces after a single oral dose, permitting the evaluation of pharmacodynamic properties. Our results define a strategy for translation of live bacterial therapeutics to treat metabolic disorders.

Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria

Tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency

Polymeric artificial cells have the potential to be used for a wide variety of therapeutic applications, such as the encapsulation of transplanted islet cells to treat diabetic patients. Recent advances in biotechnology, molecular biology, nanotechnology and polymer chemistry are now opening up further exciting possibilities in this field. However, it is also recognized that there are several key obstacles to overcome in bringing such approaches into routine clinical use. This review describes the historical development and principles behind polymeric artificial cells, the present state of the art in their therapeutic application, and the promises and challenges for the future.

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Therapeutic applications of polymeric artificial cells

Phenylketonuria (PKU), with its associated hyperphenylalaninemia (HPA) and mental retardation, is a classic genetic disease and the first to have an identified chemical cause of impaired cognitive development. Treatment from birth with a low phenylalanine diet largely prevents the deviant cognitive phenotype by ameliorating HPA and is recognized as one of the first effective treatments of a genetic disease. However, compliance with dietary treatment is difficult and when it is for life, as now recommended by an internationally used set of guidelines, is probably unrealistic. Herein we describe experiments on a mouse model using another modality for treatment of PKU compatible with better compliance using ancillary phenylalanine ammonia lyase (PAL, EC 4.3.1.5) to degrade phenylalanine, the harmful nutrient in PKU; in this treatment, PAL acts as a substitute for the enzyme phenylalanine monooxygenase (EC 1.14.16.1), which is deficient in PKU. PAL, a robust enzyme without need for a cofactor, converts phenylalanine to trans-cinnamic acid, a harmless metabolite. We describe (i) an efficient recombinant approach to produce PAL enzyme, (ii) testing of PAL in orthologous N-ethyl-N′-nitrosourea (ENU) mutant mouse strains with HPA, and (iii) proofs of principle (PAL reduces HPA)—both pharmacologic (with a clear dose–response effect vs. HPA after PAL injection) and physiologic (protected enteral PAL is significantly effective vs. HPA). These findings open another way to facilitate treatment of this classic genetic disease.

https://www.pnas.org/content/pnas/96/5/2339.full.pdf

A different approach to treatment of phenylketonuria: phenylalanine degradation with recombinant phenylalanine ammonia lyase.

Methods to microencapsulate enzyme, cells, and genetically engineered cells have been described in this article. More specific examples of enzyme encapsulation include the microencapsulation of xanthine oxidase for Lesch-Nyhan disease; phenylalanine ammonia lyase for pheny, ketonuria and microencapsulation of multienzyme systems with cofactor recycling for multistep enzyme conversions. Methods for cell encapsulation include the details for encapsulating hepatocytes for liver failure and for gene therapy. This also includes the details of a novel two-step method for encapsulation of high concentrations of smaller cells. Another new approach is the detailed method of the encapsulation of genetically engineered Escherichia coli DH5 cells for lowering urea, ammonia, and other metabolites in kidney or, liver failure and other diseases.

Procedures for microencapsulation of enzymes, cells and genetically engineered microorganisms.

Phenylalanine ammonia-lyase immobilized in microcapsules for the depletion of phenylalanine in plasma in phenylketonuric rat model.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2885%2980219-2

Phenylalanine ammonia-lyase immobilized in semipermeable microcapsules for enzyme replacement in phenylketonuria.

Oral binders remove intestinal bile acid and prevent its reabsorption and recycling thereby lowering systemic cholesterol levels. The results in this paper demonstrate the presence of another extensive enterorecirculation for amino acids. Pancreatic and other glandular secretions into the intestine contain large amounts of proteins, enzymes and polypeptides. Tryptic digestion converts these into amino acids which are then reabsorbed back into the body as they pass down the intestine. This paper shows that this forms a large enterorecirculation of amino acids between the body and intestine. The dietary protein source of amino acids is negligible when compared to the endogenous source, since this paper shows that protein-free diet did not alter the intestinal amino acid concentration. This raises the possibility of using this for the selective depletion of specific body amino acids. In this paper we use a phenylketonuria (PKU) model in rats to test the use of this hypothesis. In PKU rats, artificial cells m...

A new theory of enterorecirculation of amino acids and its use for depleting unwanted amino acids using oral enzyme-artificial cells, as in removing phenylalanine in phenylketonuria.

Intraperitoneal injections of para-Chlorophenylalanine methyl ester and Phenylalanine solution were used to produce phenylketonuria in rats. By the use of different diets and orally administered Phenylalanine ammonia lyase (PAL) loaded artificial cells, the Phenylalanine concentration gradient between the plasma and the gastro-intestinal lumen was varied. Other changes in related amino acids levels were also studied. The transport of neutral amino acids, across the gastro-intestinal membrane to the plasma, was decreased by the presence of a high concentration of Phenylalanine in the intestinal lumen. Unlike Phenylalanine free diet, oral administration of PAL loaded artificial cells to PKU rats on normal diets resulted in much lower levels of intestinal phenylalanine.

Effects of Oral Administration of Artificial Cells Immobilized Phenylalanine Ammonia-Lyase on Intestinal Amino Acids of Phenylketonuric Rats

A microcapsule for oral administration comprising an outer layer membrane which is acid resistant and retains its structure in an alkaline environment, said membrane having dispersed therein at least one enzyme corresponding to an amino acid secreted into the intestinal or derived from a protein or peptide secreted into the intestinal tract. The present invention provides a microcapsule capable of removing specific unwanted amino acids secreted into the intestinal tract.

Louis Bourget

Papers

Phenylalanine ammonia-lyase immobilized in microcapsules for the depletion of phenylalanine in plasma in phenylketonuric rat model.

Phenylalanine ammonia-lyase immobilized in semipermeable microcapsules for enzyme replacement in phenylketonuria.

A new theory of enterorecirculation of amino acids and its use for depleting unwanted amino acids using oral enzyme-artificial cells, as in removing phenylalanine in phenylketonuria.

Effects of Oral Administration of Artificial Cells Immobilized Phenylalanine Ammonia-Lyase on Intestinal Amino Acids of Phenylketonuric Rats

Microcapsule for the removal of specific amino acids through the entero-portal recirculation