Erythrocytes loaded with phenylalanine ammonia lyase (PAL) as enzymatic replacement therapy for phenylketonuria

Phenylketonuria (PKU) is an inborn error of metabolism involving the enzyme phenylalanine (L-Phe) hydroxylase (PAH) which converts L-Phe to tyrosine (L-Tyr). Untreated PKU resulting in severe and enduring neurodevelopmental disorders, can be partially prevented by an early limitation of L-Phe intake. To improve the outcome of the disease and overcome the limitations of the diet approach, enzyme substitution therapy with recombinant Anabaena variabilis Phenylalanine Ammonia Lyase (rAvPAL) has been proposed for adults with PKU. Despite the advantages, there are two problems related to the use of PAL enzyme: the first concerns its sensitivity to the acidic pH (optimal pH for PAL activity is >8) of the upper gastrointestinal tract (pH between 6 and 7.4) and its proteolytic degradation into the intestinal lumen when p.os supplied; the second, concerns the reduced half-life in blood caused by the immune activation when PAL is injected via the parenteral route, especially after repeated treatments. In order to overcome these problems, different formulations and strategies have been tested. A first attempt consisted in the binding, at a particular ratio, of PEG to lysine residues of the enzyme side chains in order to modify the protein surface and to evade the immune response, thus avoiding the premature elimination of the circulating enzyme. Although PEG system has some important features that make it suitable for pharmaceutical use, such as lack of toxicity and high solubility, this molecule itself can be immunogenic, especially upon repeated and continuous treatments. In addition, PEGylation process can modify enzyme activity compromising the therapeutic effect and require rather high costs of implementation. Indeed, PalynziqTM consisting of rAvPAL conjugated to PEG, recently approved by FDA for the treatment of PKU, may cause serious side effects including joint pain, hypersensitivity reactions due to formation of antibodies against the product as well as anaphylaxis events which require the patients to have auto-injectable epinephrine available at all times of Palynziq treatment. Considering all these drawbacks, administration through an opportune delivery system could therefore represent a valid and viable alternative to reduce the antibody response and ensure the adequate therapeutic efficacy. Particular interest has been paid on red blood cells (RBCs) thanks to the best advantages that these cells offer, making RBCs the ideal carriers for enzymes. While the pharmacological efficacy of the rAvPAL enzyme has been proved, its effectiveness in preventing the neurological impairment in early treated PKU remains to be explored. Here, we demonstrate in a pre-clinical study that weekly administrations of RBCs encapsulated with rAvPAL (rAvPAL-RBCs) from 15 to 64 post-natal days (PND) are able to normalize blood and brain L-Phe in BTBR-Pahenu2-/- mice, a murine model of the disease. In addition, behavioral (Elevated Plus Maze and Object Recognition Test), neurochemical (brain phenylalanine, tyrosine, serotonin, dopamine, and noradrenaline) and brain histological (spine density and morphology of cortical neurons, myelination) examinations were performed in treated mice and compared with untreated and wild type animals. The results demonstrated that an early treatment started at PND 15 prevents cognitive impairment, brain neurotransmitter deficit and neuropathological abnormalities commonly found in untreated PAHenu2(-/-) adult mice. No adverse events were observed. In particular, the treatment does not evoke relevant immunological reaction. Despite these promising results, the therapy failed to restore normal brain L-Tyr level, dopaminergic transmission and related motor functions. However, since serotonin metabolism, myelin synthesis and the development of cortical dendritic tree are specifically affected by early exposure to phenylalanine, the ability of rAvPAL loaded RBCs to efficiently remove L-Phe from blood, represents a worth approach not only for protecting neuropsychological functions in adult PKU subjects, but also a promising therapy to prevent the neurodevelopmental consequences of PKU in immature brain