Iijima Osamu, Miyake Koichi, Watanabe Atsushi, Miyake Noriko, Igarashi Tsutomu, Kanokoda Chizu, Nakamura-Takahashi Aki, Kinoshita Hideaki, Noguchi Taku, Abe Shinichi, Narisawa Sonoko, Millán José Luis, Okada Takashi, Shimada Takashi
1 Division of Gene Therapy, Department of Biochemistry and Molecular Biology, Research Center for Advanced Medical Technology, Nippon Medical School, Tokyo, Japan .
2 Division of Clinical Genetics, Nippon Medical School Hospital, Tokyo, Japan .
Hum Gene Ther. 2015 Dec;26(12):801-12. doi: 10.1089/hum.2015.078. Epub 2015 Nov 19.
Hypophosphatasia (HPP) is an inherited skeletal and dental disease caused by loss-of-function mutations in the gene that encodes tissue-nonspecific alkaline phosphatase (TNALP). The major symptoms of severe forms of the disease are bone defects, respiratory insufficiency, and epileptic seizures. In 2015, enzyme replacement therapy (ERT) using recombinant bone-targeted TNALP with deca-aspartate (D10) motif was approved to treat pediatric HPP patients in Japan, Canada, and Europe. However, the ERT requires repeated subcutaneous administration of the enzyme because of the short half-life in serum. In the present study, we evaluated the feasibility of neonatal ex vivo gene therapy in TNALP knockout (Akp2(-/-)) HPP mice using lentivirally transduced bone marrow cells (BMC) expressing bone-targeted TNALP in which a D10 sequence was linked to the C-terminus of soluble TNALP (TNALP-D10). The Akp2(-/-) mice usually die within 20 days because of growth failure, epileptic seizures, and hypomineralization. However, an intravenous transplantation of BMC expressing TNALP-D10 (ALP-BMC) into neonatal Akp2(-/-) mice prolonged survival of the mice with improved bone mineralization compared with untransduced BMC-transplanted Akp2(-/-) mice. The treated Akp2(-/-) mice were normal in appearance and experienced no seizures during the experimental period. The lentivirally transduced BMC were efficiently engrafted in the recipient mice and supplied TNALP-D10 continuously at a therapeutic level for at least 3 months. Moreover, TNALP-D10 overexpression did not affect multilineage reconstitution in the recipient mice. The plasma ALP activity was sustained at high levels in the treated mice, and tissue ALP activity was selectively detected on bone surfaces, not in the kidneys or other organs. No ectopic calcification was observed in the ALP-BMC-treated mice. These results indicate that lentivirally transduced BMC can serve as a reservoir for stem cell-based ERT to rescue the Akp2(-/-) phenotype. Neonatal ex vivo gene therapy thus appears to be a possible treatment option for treating severe HPP.
低磷酸酯酶症（HPP）是一种遗传性骨骼和牙齿疾病，由编码组织非特异性碱性磷酸酶（TNALP）的基因功能丧失突变引起。该疾病严重形式的主要症状是骨缺陷、呼吸功能不全和癫痫发作。2015年，使用带有十聚天冬氨酸（D10）基序的重组骨靶向TNALP的酶替代疗法（ERT）在日本、加拿大和欧洲被批准用于治疗小儿HPP患者。然而，由于该酶在血清中的半衰期较短，ERT需要反复皮下注射该酶。在本研究中，我们评估了在低磷酸酯酶症基因敲除（Akp2(-/-)）的HPP小鼠中进行新生鼠离体基因治疗的可行性，方法是使用慢病毒转导的表达骨靶向TNALP的骨髓细胞（BMC），其中D10序列连接到可溶性TNALP（TNALP-D10）的C末端。Akp2(-/-)小鼠通常因生长衰竭、癫痫发作和矿化不足在20天内死亡。然而，将表达TNALP-D10的BMC（ALP-BMC）静脉移植到新生Akp2(-/-)小鼠中，与未转导BMC移植的Akp2(-/-)小鼠相比，延长了小鼠的生存期并改善了骨矿化。接受治疗的Akp2(-/-)小鼠外观正常，在实验期间未出现癫痫发作。慢病毒转导的BMC有效地植入受体小鼠体内，并以治疗水平持续供应TNALP-D10至少3个月。此外，TNALP-D10的过表达不影响受体小鼠的多谱系重建。治疗小鼠的血浆碱性磷酸酶活性维持在高水平，并且在骨表面选择性检测到组织碱性磷酸酶活性，而在肾脏或其他器官中未检测到。在接受ALP-BMC治疗的小鼠中未观察到异位钙化。这些结果表明，慢病毒转导的BMC可以作为基于干细胞的ERT的储存库，以挽救Akp2(-/-)表型。因此，新生鼠离体基因治疗似乎是治疗严重HPP的一种可能的治疗选择。
