Enzyme replacement therapy for murine hypophosphatasia

Abstract

Introduction: Hypophosphatasia (HPP) is the inborn error of metabolism that features rickets or osteomalacia caused by loss-of-function mutation(s) within the gene that encodes the tissue-nonspecific isozyme of alkaline phosphatase (TNALP). Consequently, natural substrates for this ectoenzyme accumulate extracellulary including inorganic pyrophosphate (PPi), an inhibitor of mineralization, and pyridoxal 5'-phosphate (PLP), a co-factor form of vitamin B6. Babies with the infantile form of HPP often die with severe rickets and sometimes hypercalcemia and vitamin B6-dependent seizures. There is no established medical treatment.

Materials and methods: Human TNALP was bioengineered with the C terminus extended by the Fc region of human IgG for one-step purification and a deca-aspartate sequence (D10) for targeting to mineralizing tissue (sALP-FcD10). TNALP-null mice (Akp2-/-), an excellent model for infantile HPP, were treated from birth using sALP-FcD10. Short-term and long-term efficacy studies consisted of once daily subcutaneous injections of 1, 2, or 8.2 mg/kg sALP-FcD10 for 15, 19, and 15 or 52 days, respectively. We assessed survival and growth rates, circulating levels of sALP-FcD10 activity, calcium, PPi, and pyridoxal, as well as skeletal and dental manifestations using radiography, microCT, and histomorphometry.

Results: Akp2-/- mice receiving high-dose sALP-FcD10 grew normally and appeared well without skeletal or dental disease or epilepsy. Plasma calcium, PPi, and pyridoxal concentrations remained in their normal ranges. We found no evidence of significant skeletal or dental disease.

Conclusions: Enzyme replacement using a bone-targeted, recombinant form of human TNALP prevents infantile HPP in Akp2-/- mice.

Figures

FIG. 1

Purification and properties of recombinant sALP-FcD10 and pharmacokinetic and tissue distribution studies. (A) SDS-PAGE of purified sALP-FcD10. Protein purified by Protein A-Sepharose affinity chromatography was analyzed by SDS-PAGE and bands were stained with Sypro Ruby. sALP-FcD10 migrated as the major species with an apparent molecular mass of ∼90,000 Da under reducing conditions (Red) and ∼200,000 Da under nonreducing, native conditions (Nat). (B) Characterization of sALP-FcD10 by molecular sieve chromatography under nondenaturing conditions. Purified sALP-FcD10 protein (2 mg) was resolved on a calibrated column of Sephacryl S-300. The principal form of sALP-FcD10 (Peak 3), consisting of 80% of the total material deposited on the column, eluted with a molecular mass of 370,000 Da consistent with a tetrameric structure. When analyzed by SDS-PAGE in the presence of dithiothreitol (DTT), the material in peak 3 migrated with an apparent molecular mass of a monomer. In the absence of DTT, the protein migrated with the mobility of a dimer. (C) Concentrations of radiolabeled sALP-FcD10 in serum, tibia, and muscle, expressed as micrograms per gram tissue (wet weight), after a single intravenous bolus of 5 mg/kg in adult WT mice (n = 3). (D) Serum concentrations of radiolabeled sALP-FcD10 as a function of time after a single subcutaneous injection of 3.7 mg/kg in 1-day-old WT mice (n = 3). (E) Histochemical staining for ALP activity in the long bones of sALP-FcD10–treated Akp2−/− mice. Proximal tibia with EzRT compared with an age-matched untreated Akp2−/− mouse. Arrows show areas of ALP activity staining. Bars: 100 μm.

FIG. 2

Short-term, high-dose (8.2 mg/kg) EzRT efficacy studies in Akp2−/− mice. (A) Plasma concentrations of ALP activity. (B) Growth curves of Akp2−/− mice given vehicle (n = 18) or sALP-FcD10 (n = 19) and nontreated WT mice (n = 18). (C) X-ray images of feet, rib cages, and hind limbs of Akp2−/− mice (16 days) and a Faxitron image distribution table. Severely (s) affected mice had absence of digital bones (phalanges) and secondary ossification centers. Moderately (m) affected mice had abnormal secondary ossification centers, but all digital bones were present. Healthy (h) WT mice had all bony structures present with normal architecture. Radiographic images of the hind limbs were similarly classified as abnormal if evidence of acute or chronic fractures was present or healthy in the absence of any abnormality.

FIG. 3

Preservation of Akp2−/− tooth and alveolar bone architecture by EzRT. Mandibles from 16-day-old mice (WT, sALP-FcD10-treated Akp2−/−, and vehicle-injected Akp2−/−) were cut into segments containing the first molar, the underlying incisor, and the surrounding alveolar bone. Tooth type and treatment are as indicated. Note the incisor from a vehicle-injected Akp2−/− mouse showing only partial mineralization of the dentin. Extensive regions of unmineralized crown analog dentin (open arrow) and unmineralized root analog dentin (arrow) are present. Likewise, the surrounding alveolar bone also shows regions of unmineralized bone matrix (asterisk). sALP-FcD10 treatment of Akp2−/− mice preserves complete mineralization of all incisor tooth tissues and the surrounding alveolar bone, such that no mineralization differences are seen between the incisor teeth and bone of the EzRT mice compared with WT mice. Note also the molar from a vehicle-injected Akp2−/− mouse showing only partial mineralization of the dentin. Extensive regions of unmineralized root dentin (arrows) are present, as are regions of unmineralized alveolar bone matrix (asterisk). EzRT maintains complete mineralization of all molar dentin as well as the surrounding alveolar bone such that no mineralization differences are seen between the molar teeth and bone of the treated mice compared with WT mice. Magnification bar = 100 μm. All images were taken at the same magnification.

FIG. 4

Long-term (52 days), high-dose (8.2 mg/kg) EzRT efficacy studies in Akp2−/− mice. (A) Long-term survival of EzRT mice contrasts with precipitous, early demise of the vehicle-treated group. (B) Normal appearance of an EzRT mouse compared with a stunted, vehicle-treated animal. (C) Plasma ALP concentration in untreated and treated Akp2−/− mice and WT controls. (D) X-ray image of the hind paw of an 18-day-old untreated Akp2−/− mouse showing absence of secondary ossification centers (arrow). In contrast, note the normal appearances of the hind paws of 46- and 52-day-old Akp2−/− mice receiving EzRT.