Prolonged survival and phenotypic correction of Akp2(-/-) hypophosphatasia mice by lentiviral gene therapy

Abstract

Hypophosphatasia (HPP) is an inherited systemic skeletal disease caused by mutations in the gene encoding the tissue-nonspecific alkaline phosphatase (TNALP) isozyme. The clinical severity of HPP varies widely, with symptoms including rickets and osteomalacia. TNALP knockout (Akp2(-/-)) mice phenotypically mimic the severe infantile form of HPP; that is, TNALP-deficient mice are born with a normal appearance but die by 20 days of age owing to growth failure, hypomineralization, and epileptic seizures. In this study, a lentiviral vector expressing a bone-targeted form of TNALP was injected into the jugular vein of newborn Akp2(-/-) mice. We found that alkaline phosphatase activity in the plasma of treated Akp2(-/-) mice increased and remained at high levels throughout the life of the animals. The treated Akp2(-/-) mice survived for more than 10 months and demonstrated normal physical activity and a healthy appearance. Epileptic seizures were completely inhibited in the treated Akp2(-/-) mice, and X-ray examination of the skeleton showed that mineralization was significantly improved by the gene therapy. These results show that severe infantile HPP in TNALP knockout mice can be treated with a single injection of lentiviral vector during the neonatal period.

Fig. 1

Lentiviral-mediated gene therapy of Akp2^−/− hypophosphatasic (HPP) mice. (A) Schematic diagram of HIV-TNALP-D10 lentiviral vector. LTR = long terminal repeat; MSCVU3 = U3 region of the LTR promoter of murine stem cell virus; WPRE = woodchuck hepatitis virus posttranscriptional regulatory element; INS = chicken β-globin hypersensitivity site 4 insulator; cppt-cts = central polypurine tract–central termination sequence; RRE = reverse responsive element. (B) Growth curves of untreated HPP mice (n = 7), treated HPP mice (n = 6), and WT (n = 4) and HET (n = 9) mice. The body weights of untreated HPP mice were recorded until spontaneous death. The weights of WT and HET (total n = 13) mice are presented as the average ± SD. (C) The comparison of average body weights of treated HPP mice (male, n = 3; female, n = 3) and WT/HET littermates (male, n = 8; female, n = 8 to 10). ^*p < .05; ^***p < .001. (D) The survival curves of treated (n = 7) and untreated (n = 12) HPP mice. (E) Concentration of plasma ALP in the treated (n = 6) and untreated (n = 5) HPP mice and HET (n = 15 to 21) and WT (n = 6-9) controls. ^**p < .01 versus the WT group; ^***p < .001 versus the WT group. (F) Distribution of lentiviral vector. The copy numbers of the vector genome in the organs was determined by qPCR with HIV-TNALP-D10 injected WT mice. Data are presented as mean ± SEM (n = 4).

Fig. 2

X-ray images of the feet. Secondary ossification centers in the hind paws were absent in untreated HPP mice but were detectable in the treated mice at 18 days after birth. No differences in skeletal mineralization were observed between treated long survivors and WT mice at 100 days of age.

Fig. 3

Histochemical staining of ALP activity in the tibias. ALP activity was detected in WT (A) but not HPP mice (B) at 15 days after birth. Following treatment with lentiviral vector, (C) ALP activity was detected on the surface of the endosteal bone at 18 days after birth. (D) Magnified image of the square in panel C. Bars = 1 mm.