Erythrocytes encapsulated with phenylalanine hydroxylase exhibit improved pharmacokinetics and lowered plasma phenylalanine levels in normal mice.

Enzyme replacement therapy is often hampered by the rapid clearance and degradation of the administered enzyme, limiting its efficacy and requiring frequent dosing. Encapsulation of therapeutic molecules into red blood cells (RBCs) is a clinically proven approach to improve the pharmacokinetics and efficacy of biologics and small molecule drugs. Here we evaluated the ability of RBCs encapsulated with phenylalanine hydroxylase (PAH) to metabolize phenylalanine (Phe) from the blood and confer sustained enzymatic activity in the circulation. Significant quantities of PAH were successfully encapsulated within murine RBCs (PAH-RBCs) with minimal loss of endogenous hemoglobin. While intravenously administered free PAH enzyme was rapidly eliminated from the blood within a few hours, PAH-RBCs persisted in the circulation for at least 10days. A single injection of PAH-RBCs was able to decrease Phe levels by nearly 80% in normal mice. These results demonstrate the ability of enzyme-loaded RBCs to metabolize circulating amino acids and highlight the potential to treat disorders of amino acid metabolism.

The activity of wild-type and mutant phenylalanine hydroxylase and its regulation by phenylalanine and tetrahydrobiopterin at physiological and pathological concentrations: an isothermal titration calorimetry study.

The activity of phenylalanine hydroxylase (PAH) is regulated by the levels of both the substrate (L-Phe) and the natural cofactor (6R)-tetrahydrobiopterin (BH4). It has recently been observed that many PAH mutants associated with BH4-responsive phenylketonuria display abnormal kinetic and regulatory properties as shown by standard kinetic analyses. In this work, we have developed a high-sensitive and high-throughput activity assay based on isothermal titration calorimetry (ITC) in order to study the kinetic properties of wild-type PAH (wt-PAH) and the BH4-responsive c.204A>T (p.R68S) mutant at physiological and superphysiological concentrations of L-Phe and BH4. Compared to wt-PAH, the p.R68S mutant showed reduced apparent and equilibrium binding affinity for the natural cofactor and increased affinity and non-cooperative response for L-Phe, together with a strong substrate inhibition that is alleviated at high BH4 concentrations. For both wt-PAH and mutant, the apparent affinity for BH4 decreases at increasing L-Phe concentrations, and the affinity for the substrate also depends on the cofactor concentration. Our results indicate that the activity landscape for wt and mutant enzymes is more complex than expected from standard kinetic analyses and highlight the applicability of this ITC-based assay to characterize the activity and regulation of PAH at a wide range of substrate and cofactor concentrations. Moreover, the results aid to understand the activity dynamics of wild-type and mutant PAH under physiological and pathological conditions, as well as BH4-responsiveness in certain PKU mutations.