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Abstract
Phenylketonuria (PKU) is a genetic metabolic disease in which the decrease or loss of phenylalanine hydroxylase (PAH) activity results in elevated, neurotoxic levels of phenylalanine (Phe). Due to many obstacles, PAH enzyme replacement therapy is not currently an option. Treatment of PKU with an alternative enzyme, phenylalanine ammonia lyase (PAL), was first proposed in the 1970s. However, issues regarding immunogenicity, enzyme production and mode of delivery needed to be overcome. Through the evaluation of PAL enzymes from multiple species, three potential PAL enzymes from yeast and cyanobacteria were chosen for evaluation of their therapeutic potential. The addition of polyethylene glycol (PEG, MW = 20,000), at a particular ratio to modify the protein surface, attenuated immunogenicity in an animal model of PKU. All three PEGylated PAL candidates showed efficacy in a mouse model of PKU (BTBR Pahenu2) upon subcutaneous injection. However, only PEGylated Anabaena variabilis (Av) PAL-treated mice demonstrated sustained low Phe levels with weekly injection and was the only PAL evaluated that maintained full enzymatic activity upon PEGylation. A PEGylated recombinant double mutant version of AvPAL (Cys503Ser/Cys565Ser), rAvPAL-PEG, was selected for drug development based on its positive pharmacodynamic profile and favorable expression titers. PEGylation was shown to be critical for rAvPAL-PEG efficacy as under PEGylated rAvPAL had a lower pharmacodynamic effect. rAvPAL and rAvPAL-PEG had poor stability at 4°C. L-Phe and trans-cinnamate were identified as activity stabilizing excipients. rAvPAL-PEG is currently in Phase 3 clinical trials to assess efficacy in PKU patients.
Highlights
Phenylketonuria (PKU; OMIM 261600) is an autosomal recessive disease, which results in a toxic excess of Phenylalanine (Phe) due to the loss of function of phenylalanine hydroxylase (PAH; EC 1.14.16.1) with an incidence of 1:15,000 live births [1]
Recombinant NpPAL, RtPAL(R91K), AvPALWT, AvPAL containing mutations to prevent aggregation, C503S/C565S, as well as 91 phenylalanine ammonia lyase (PAL) from unidentified microbial origin were expressed from a pIBEX7 plasmid in the E. coli strains BLR or BL21(DE3) (EMD Millipore, Billerica, MA) and purified by hydrophobic interacting chromatography followed by anion exchange chromatography [20, 30]
Results rAvPAL(C503S/C565S) retains activity upon PEGylation In Wang, et al, we described the structural and biochemical characteristics of rAvPALWT and a mutant form of rAvPAL with two mutated cysteines, Cys503 and Cys565 [32]. rAvPAL (C503S/C565S), hereafter referred to as rAvPAL, had similar specific activity and kcat/Km to rAvPALWT (Table 1, Wang, et al, 2008). rAvPAL expression as soluble protein in E. coli was greater than both rNpPAL and rRtPAL (Table 1). rAvPAL (WT and mutant) titers ranged
Summary
Phenylketonuria (PKU; OMIM 261600) is an autosomal recessive disease, which results in a toxic excess of Phenylalanine (Phe) due to the loss of function of phenylalanine hydroxylase (PAH; EC 1.14.16.1) with an incidence of 1:15,000 live births [1]. Since the 1960s, newborns have been tested for PAH enzyme deficiency and if positive, prescribed a low Phe diet [3] Compliance to such a restricted diet is low and until recently remained the only treatment [4, 5]. Not all PKU patients are completely deficient for PAH activity and a subset of patients respond to oral (6R)-L-erythro-5,6,7,8-tetrahydropiopterin (6R-BH4, sapropterin), the PAH cofactor [6, 7]. This subset comprises 30–50% of the PKU patient population, and many of those that respond to therapy continue to have elevated Phe levels, albeit lower than without sapropterin treatment [8]
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