Achondroplasia (ACH), the most common form of dwarfism, is an inherited autosomal-dominant chondrodysplasia caused by a gain-of-function mutation in fibroblast-growth-factor-receptor 3 (FGFR3). C-type natriuretic peptide (CNP) antagonizes FGFR3 downstream signaling by inhibiting the pathway of mitogen-activated protein kinase (MAPK). Here, we report the pharmacological activity of a 39 amino acid CNP analog (BMN 111) with an extended plasma half-life due to its resistance to neutral-endopeptidase (NEP) digestion. In ACH human growth-plate chondrocytes, we demonstrated a decrease in the phosphorylation of extracellular-signal-regulated kinases 1 and 2, confirming that this CNP analog inhibits fibroblast-growth-factor-mediated MAPK activation. Concomitantly, we analyzed the phenotype of Fgfr3(Y367C/+) mice and showed the presence of ACH-related clinical features in this mouse model. We found that in Fgfr3(Y367C/+) mice, treatment with this CNP analog led to a significant recovery of bone growth. We observed an increase in the axial and appendicular skeleton lengths, and improvements in dwarfism-related clinical features included flattening of the skull, reduced crossbite, straightening of the tibias and femurs, and correction of the growth-plate defect. Thus, our results provide the proof of concept that BMN 111, a NEP-resistant CNP analog, might benefit individuals with ACH and hypochondroplasia.

We have recently observed promising success in a mouse model at treating the metabolic disorder phenylketonuria (PKU) with phenylalanine ammonia lyase (PAL) from R. toruloides and A. variabilis. Both molecules, however, required further optimization in order to overcome problems with protease susceptibility, thermal stability and aggregation. We reduced aggregation of the A. variabilis PAL by mutating two surface cysteine residues (C503 and C565) to serines. Here we report the structural and biochemical characterization of the A. variabilis PAL C503S/C565S double mutant. Unlike previously published PAL structures, significant electron density is observed for the two active site loops in the A. variabilis C503S/C565S double mutant, yielding a complete view of the active site. Docking studies and NHS-biotin binding studies support a proposed mechanism in which the amino group of the phenylalanine substrate is attacked directly by the 4-methylideneimidazole-5-one (MIO) prosthetic group. We propose a helix-to-loop conformational switch in the helices flanking the inner active site loop that regulates accessibility of the active site. Differences in loop stability among PAL homologs may explain the observed variation in enzyme efficiency despite the highly conserved structure of the active site. A. variabilis C503S/C565S PAL is shown to be both more thermally stable and more resistant to proteolytic cleavage than R. toruloides PAL. Additional increases in thermal stability and protease resistance upon ligand binding may be due to enhanced interactions among the residues of the active site, possibly locking the active site structure in place and stabilizing the tetramer. Examination of the A. variabilis C503S/C565S PAL structure combined with analysis of its physical properties provides a structural basis for further engineering of residues that could result in a better therapeutic molecule.

Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is a lysosomal storage disorder caused by deficiency of N-acetylgalactosamine-6-sulfatase (GALNS), an enzyme that degrades keratan sulfate (KS). Currently no therapy for MPS IVA is available. We produced recombinant human (rh)GALNS as a potential enzyme replacement therapy for MPS IVA. Chinese hamster ovary cells stably overexpressing GALNS and sulfatase modifying factor-1 were used to produce active (∼2 U/mg) and pure (≥97%) rhGALNS. The recombinant enzyme was phosphorylated and was dose-dependently taken up by mannose-6-phosphate receptor (Kuptake = 2.5 nM), thereby restoring enzyme activity in MPS IVA fibroblasts. In the absence of an animal model with a skeletal phenotype, we established chondrocytes isolated from two MPS IVA patients as a disease model in vitro. MPS IVA chondrocyte GALNS activity was not detectable and the cells exhibited KS storage up to 11-fold higher than unaffected chondrocytes. MPS IVA chondrocytes internalized rhGALNS into lysosomes, resulting in normalization of enzyme activity and decrease in KS storage. rhGALNS treatment also modulated gene expression, increasing expression of chondrogenic genes Collagen II, Collagen X, Aggrecan and Sox9 and decreasing abnormal expression of Collagen I. Intravenous administration of rhGALNS resulted in biodistribution throughout all layers of the heart valve and the entire thickness of the growth plate in wild-type mice. We show that enzyme replacement therapy with recombinant human GALNS results in clearance of keratan sulfate accumulation, and that such treatment ameliorates aberrant gene expression in human chondrocytes in vitro. Penetration of the therapeutic enzyme throughout poorly vascularized, but clinically relevant tissues, including growth plate cartilage and heart valve, as well as macrophages and hepatocytes in wild-type mouse, further supports development of rhGALNS as enzyme replacement therapy for MPS IVA.

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.

Enzyme replacement therapy for lysosomal storage disorders depends on efficient uptake of recombinant enzyme into the tissues of patients. This uptake is mediated by oligosaccharide receptors including the cation-independent mannose 6-phosphate receptor and the mannose receptor. We have sought to exploit alternative receptor systems that are independent of glycosylation but allow for efficient delivery to the lysosome. Fusions of the human lysosomal enzymes ␣-L-iduronidase or acid ␣-glucosidase with the receptor-associated protein were efficiently endocytosed by lysosomal storage disorder patient fibroblasts, rat C6 glioma cells, mouse C2C12 myoblasts, and recombinant Chinese hamster ovary cells expressing individual members of the low-density lipoprotein receptor family. Uptake of the fusions exceeded that of phosphorylated enzyme in all cases, often by an order of magnitude or greater. Uptake was specifically mediated by members of the low-density lipoprotein receptor protein family and was followed by delivery of the fusions to the lysosome. The advantages of the lipoprotein receptor system over oligosaccharide receptor systems include more efficient cellular delivery and the potential for transcytosis of ligands across tight endothelia, including the blood-brain barrier.Enzyme replacement therapy for lysosomal storage disorders relies on the efficient delivery of recombinant enzyme to all of the affected tissues of the patient (1). Uptake of enzyme from the blood following intravenous administration requires specific oligosaccharides on the enzyme itself and corresponding oligosaccharide receptors on target cells. Examples include the binding of phosphorylated high-mannose oligosaccharides on ␣-L-iduronidase by the cation-independent mannose 6-phosphate receptor (MPR) 1 and binding of high-mannose oligosaccharides on glucocerebrosidase by the mannose receptor (2, 3). The former system is the basis for treatment of patients with Hurler syndrome, the latter for Gaucher syndrome. Factors that limit uptake by these systems include the extent to which the recombinant enzyme is modified with the necessary oligosaccharides and the density of the oligosaccharide receptors on different tissues. The absence of quality control mechanisms for oligosaccharide modifications in the secretory pathway can lead to poorly modified recombinant enzymes. This deficiency has been addressed, with varying success, by post-secretory modification of recombinant enzymes with glycosidases or glycosyltransferases (4). Another solution to the problem is to utilize alternative uptake mechanisms that rely on proteinbased, rather than oligosaccharide-based, targeting determinants. Some studies have focused on the use of small, basic peptides, like human immunodeficiency virus TAT, for this purpose (5, 6). Others have demonstrated the feasibility of this approach using insulin-like growth factor-2, a peptide ligand for the MPR (7). The low density lipoprotein receptor (LDLR) family is one of the most widely distributed and intensively utili...