Background: Phenylketonuria (PKU) is a congenital metabolic disorder caused by the deficiency or mutation of phenylalanine hydroxylase (PAH). Patients with PKU must restrict their intake of phenylalanine and are therefore unable to consume most protein-containing foods. In recent years, enzyme replacement therapy for PKU has been approved; however, patients are required to self-inject the drug on a daily basis. To reduce treatment frequency and improve patient convenience, we are developing an erythrocyte-based enzyme delivery system by encapsulating PAH or metabolically similar enzymes into red blood cells.

Objective: The aim of this study was to evaluate the enzymatic activity of recombinant enzymes both outside and inside red blood cells, with the goal of assessing the feasibility of a novel treatment for PKU.

Materials and Methods: Red blood cells were isolated from the blood of 8-week-old Wistar rats. As metabolic enzymes, recombinant human PAH and phenylalanine ammonia lyase (PAL) derived from cyanobacteria were used. A proprietary hypotonic loading method was employed for enzyme encapsulation.

Results: The enzymatic activity of human PAH varied depending on the position of purification tags and the enzyme concentration. Although a major functional domain of the lncRNA reportedly involved in PAH activation was added, no significant change in enzymatic activity was observed. PAH activity could not be detected within red blood cells. In contrast, PAL exhibited high enzymatic activity and successfully reduced phenylalanine levels even inside erythrocytes.

Discussion: From the standpoint of immunogenicity, human PAH would be the ideal enzyme; however, modifications such as glycosylation may be required to enhance its stability and function. On the other hand, PAL demonstrated greater practicality under current conditions. Future studies will involve the administration of PAL-loaded erythrocytes into animal models to determine whether phenylalanine in the bloodstream can be effectively metabolized.